Method for producing polyisoprenoid, transformed plant, method for producing pneumatic tire and method for producing rubber product

ABSTRACT

Provided is a method for producing a polyisoprenoid, which can increase natural rubber production by enhancing the rubber synthesis activity of rubber particles. The present invention provides methods for producing a polyisoprenoid using a gene coding for a cis-prenyltransferase (CPT) family protein, a gene coding for a Nogo-B receptor (NgBR) family protein and a gene coding for a rubber elongation factor (REF) family protein, specifically a method for producing a polyisoprenoid in vitro using rubber particles bound to proteins coded for by these genes, and a method for producing a polyisoprenoid in vivo using a recombinant organism (plant) having these genes introduced therein.

TECHNICAL FIELD

The present invention relates to a method for producing apolyisoprenoid, a transformed plant, a method for producing a pneumatictire, and a method for producing a rubber product.

BACKGROUND ART

Nowadays natural rubber (one example of polyisoprenoids) for use inindustrial rubber products are obtained by cultivating rubber-producingplants, such as para rubber tree (Hevea brasiliensis) belonging to thefamily Euphorbiaceae, or Indian rubber tree (Ficus elastica) belongingto the family Moraceae, to biosynthesize natural rubber by thelactiferous cells of the plants, and harvesting the natural rubber byhand from the plants.

At present, Hevea brasiliensis is practically the only one source ofnatural rubber for industrial rubber products. Hevea brasiliensis is aplant that can grow only in limited areas such as in Southeast Asia andSouth America. Conventionally, natural rubber production has beenincreased by applying ethephon or methyl jasmonate to Hevea brasiliensistrees to induce lactiferous duct formation. Moreover, Hevea brasiliensisrequires about seven years from planting to mature enough for rubberextraction, and the period during which natural rubber can be extractedis limited to 20 to 30 years. Although more natural rubber is expectedto be demanded mainly by developing countries in years to come, for thereason mentioned above it is difficult to greatly increase theproduction of natural rubber using Hevea brasiliensis. Depletion ofnatural rubber sources is therefore of concern, and there are needs forstable natural rubber sources other than mature Hevea brasiliensis andfor improvement in productivity of natural rubber from Heveabrasiliensis.

Natural rubber has a cis-1,4-polyisoprene structure formed mainly ofisopentenyl diphosphate (IPP) units, and the nature of this structuresuggests that cis-prenyltransferase (CPT) is involved in natural rubberbiosynthesis. For example, several CPTs are found in Hevea brasiliensis,including Hevea rubber transferase 1 (HRT1) and Hevea rubber transferase2 (HRT2) (see for example Non Patent Literatures 1 and 2). It is alsoknown that rubber synthesis can be reduced in the dandelion speciesTaraxacum brevicorniculatum by suppressing CPT expression (see forexample Non Patent Literature 3).

Previous studies of proteins associated with natural rubber biosynthesishave focused on rubber elongation factor (REF) and small rubber particleprotein (SRPP) (see for example Non Patent Literatures 4 and 5).However, the associations between these proteins and CPT are notcompletely understood.

It has also been suggested that Nogo-B receptor (NgBR) is involved indolichol biosynthesis by a human CPT (see for example Non PatentLiterature 6).

Methods have been studied for increasing the production of naturalrubber in Hevea brasiliensis, but since the rubber synthesis mechanismof Hevea brasiliensis is not completely understood, Hevea brasiliensisvariants have been proposed which have been genetically modified toexpress and enhance genes of the known monomer (isopentenyl diphosphate)synthesis pathways (mevalonate (MVA) pathway and non-mevalonate (MEP)pathway) (see for example Patent Literature 1).

CITATION LIST Patent Literature

-   Patent Literature 1: JP 2005-500840 T

Non Patent Literature

-   Non Patent Literature 1: Rahaman et al., BMC Genomics, 2013, vol. 14-   Non Patent Literature 2: Asawatreratanakul et al., European Journal    of Biochemistry, 2003, vol. 270, pp. 4671-4680-   Non Patent Literature 3: Post et al., Plant Physiology, 2012, vol.    158, pp. 1406-1417-   Non Patent Literature 4: Hillebrand et al., PLoS ONE, 2012, vol. 7-   Non Patent Literature 5: Priya et al., Plant Cell Reports, 2007,    vol. 26, pp. 1833-1838-   Non Patent Literature 6: K. D. Harrison et al., The EMBO Journal,    2011, vol. 30, pp. 2490-2500

SUMMARY OF INVENTION Technical Problem

As described above, there are needs for development of stable naturalrubber sources other than mature Hevea brasiliensis and for improvementin productivity of natural rubber from Hevea brasiliensis, but atpresent, the biosynthesis mechanism of natural rubber, and particularlythe regulatory mechanism remains largely unclear, and there is stillmuch room for improvement to greatly increase natural rubber production.

Although methods have been studied for increasing the production ofnatural rubber in Hevea brasiliensis as described above, it is knownthat ethephon, methyl jasmonate and other chemicals need to be appliedto Hevea brasiliensis trees continuously, but long-term application ofsuch chemicals may damage the trees. For example, it is known that barksplitting is more likely to occur when ethephon is applied to the trunkof Hevea brasiliensis trees for a long period of time.

Moreover, even when the genes of the monomer (isopentenyl diphosphate)synthesis pathways (mevalonate (MVA) pathway and non-mevalonate (MEP)pathway) are expressed and enhanced, since the monomer materialisopentenyl diphosphate (IPP) has uses apart from a raw material ofnatural rubber, the increase in IPP production may not lead to increasednatural rubber production.

Thus, at present the biosynthesis mechanism of natural rubber, andparticularly the regulatory mechanism remains largely unclear, and thereis still much room for improvement to greatly increase natural rubberproduction.

In this context, one possible approach to solving these problems is tostabilize and enhance the activity of CPT in natural rubber biosynthesisin order to increase natural rubber production.

It is an object of the present invention to resolve these problems andprovide a method for producing a polyisoprenoid, which can increasenatural rubber production by enhancing the rubber synthesis activity ofrubber particles in vitro.

It is another object of the present invention to resolve these problemsand provide a method for producing a polyisoprenoid, which can increasenatural rubber production by producing a transformed plant with enhancedrubber synthesis activity.

Solution to Problem

The present invention relates to a method for producing apolyisoprenoid, the method including a step of binding a proteinexpressed by a gene coding for a cis-prenyltransferase (CPT) familyprotein, a protein expressed by a gene coding for a Nogo-B receptor(NgBR) family protein, and a protein expressed by a gene coding for arubber elongation factor (REF) family protein to rubber particles invitro. This invention is hereinafter called the first aspect of thepresent invention, and is also referred to simply as the firstinvention.

At least one selected from the group consisting of the gene coding for acis-prenyltransferase (CPT) family protein, the gene coding for a Nogo-Breceptor (NgBR) family protein, and the gene coding for a rubberelongation factor (REF) family protein is preferably derived from aplant.

At least one selected from the group consisting of the gene coding for acis-prenyltransferase (CPT) family protein, the gene coding for a Nogo-Breceptor (NgBR) family protein, and the gene coding for a rubberelongation factor (REF) family protein is preferably derived from Heveabrasiliensis.

The binding step preferably includes performing protein synthesis in thepresence of both rubber particles and a cell-free protein synthesissolution containing an mRNA coding for a cis-prenyltransferase (CPT)family protein, an mRNA coding for a Nogo-B receptor (NgBR) familyprotein, and an mRNA coding for a rubber elongation factor (REF) familyprotein, to bind the CPT family protein, the NgBR family protein, andthe REF family protein to the rubber particles.

The cell-free protein synthesis solution preferably contains a germextract.

The germ extract is preferably derived from wheat.

The rubber particles are preferably present in the cell-free proteinsynthesis solution at a concentration of 5 to 50 g/L.

The first invention also relates to a method for producing a pneumatictire, the method including the steps of: kneading a polyisoprenoidproduced by the method for producing a polyisoprenoid of the firstinvention with an additive to obtain a kneaded mixture; building a green(or raw) tire from the kneaded mixture; and vulcanizing the green tire.

The first invention also relates to a method for producing a rubberproduct, the method including the steps of: kneading a polyisoprenoidproduced by the method for producing a polyisoprenoid of the firstinvention with an additive to obtain a kneaded mixture; forming a rawrubber product from the kneaded mixture; and vulcanizing the raw rubberproduct.

The present invention also relates to a method for producing apolyisoprenoid, the method including producing a polyisoprenoid in atransformed plant produced by introducing a gene coding for acis-prenyltransferase (CPT) family protein, a gene coding for a Nogo-Breceptor (NgBR) family protein, and a gene coding for a rubberelongation factor (REF) family protein into a plant to allow the plantto express the CPT family protein, the NgBR family protein, and the REFfamily protein. This invention is hereinafter called the second aspectof the present invention, and is also referred to simply as the secondinvention.

At least one selected from the group consisting of the gene coding for acis-prenyltransferase (CPT) family protein, the gene coding for a Nogo-Breceptor (NgBR) family protein, and the gene coding for a rubberelongation factor (REF) family protein is preferably derived from aplant.

At least one selected from the group consisting of the gene coding for acis-prenyltransferase (CPT) family protein, the gene coding for a Nogo-Breceptor (NgBR) family protein, and the gene coding for a rubberelongation factor (REF) family protein is preferably derived from Heveabrasiliensis.

The gene coding for a cis-prenyltransferase (CPT) family protein ispreferably the following DNA [1] or [2]:

[1] a DNA having the nucleotide sequence of SEQ ID NO:1; or

[2] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:1, and codes for a protein having an enzyme activity that catalyzes areaction of cis-chain elongation of an isoprenoid compound.

The gene coding for a Nogo-B receptor (NgBR) family protein ispreferably the following DNA [3] or [4]:

[3] a DNA having the nucleotide sequence of SEQ ID NO:3; or

[4] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:3, and codes for a protein having the functions of binding to amembrane via one or more transmembrane domains on the N-terminal side ofthe protein, and interacting with another protein on the C-terminal sidethereof.

The gene coding for a rubber elongation factor (REF) family protein ispreferably the following DNA [5] or [6]:

[5] a DNA having the nucleotide sequence of SEQ ID NO:5; or

[6] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:5, and codes for a rubber particle-associated protein that is boundto rubber particles in latex.

The second invention also relates to a transformed plant, produced byintroducing a gene coding for a cis-prenyltransferase (CPT) familyprotein, a gene coding for a Nogo-B receptor (NgBR) family protein, anda gene coding for a rubber elongation factor (REF) family protein into aplant to allow the plant to express the CPT family protein, the NgBRfamily protein, and the REF family protein.

At least one selected from the group consisting of the gene coding for acis-prenyltransferase (CPT) family protein, the gene coding for a Nogo-Breceptor (NgBR) family protein, and the gene coding for a rubberelongation factor (REF) family protein is preferably derived from aplant.

At least one selected from the group consisting of the gene coding for acis-prenyltransferase (CPT) family protein, the gene coding for a Nogo-Breceptor (NgBR) family protein, and the gene coding for a rubberelongation factor (REF) family protein is preferably derived from Heveabrasiliensis.

The gene coding for a cis-prenyltransferase (CPT) family protein ispreferably the following DNA [1] or [2]:

[1] a DNA having the nucleotide sequence of SEQ ID NO:1; or

[2] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:1, and codes for a protein having an enzyme activity that catalyzes areaction of cis-chain elongation of an isoprenoid compound.

The gene coding for a Nogo-B receptor (NgBR) family protein ispreferably the following DNA [3] or [4]:

[3] a DNA having the nucleotide sequence of SEQ ID NO:3; or

[4] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:3, and codes for a protein having the functions of binding to amembrane via one or more transmembrane domains on the N-terminal side ofthe protein, and interacting with another protein on the C-terminal sidethereof.

The gene coding for a rubber elongation factor (REF) family protein ispreferably the following DNA [5] or [6]:

[5] a DNA having the nucleotide sequence of SEQ ID NO:5; or

[6] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:5, and codes for a rubber particle-associated protein that is boundto rubber particles in latex.

The second invention also relates to a method for producing a pneumatictire, the method including the steps of: kneading a polyisoprenoidproduced by the method for producing a polyisoprenoid of the secondinvention with an additive to obtain a kneaded mixture; building a greentire from the kneaded mixture; and vulcanizing the green tire.

The second invention also relates to a method for producing a rubberproduct, the method including the steps of: kneading a polyisoprenoidproduced by the method for producing a polyisoprenoid of the secondinvention with an additive to obtain a kneaded mixture; forming a rawrubber product from the kneaded mixture; and vulcanizing the raw rubberproduct.

Advantageous Effects of Invention

The method for producing a polyisoprenoid of the first inventionincludes a step of binding a protein expressed by a gene coding for acis-prenyltransferase (CPT) family protein, a protein expressed by agene coding for a Nogo-B receptor (NgBR) family protein, and a proteinexpressed by a gene coding for a rubber elongation factor (REF) familyprotein to rubber particles in vitro. Binding the CPT family protein,NgBR family protein, and REF family protein to rubber particles isexpected to stabilize and enhance the activity of the CPT familyprotein, thereby enhancing the rubber synthesis activity of rubberparticles. Thus, it is possible to produce rubber more efficiently inreaction vessels (e.g. test tubes, industrial plants).

The method for producing a pneumatic tire of the first inventionincludes the steps of: kneading a polyisoprenoid produced by the methodfor producing a polyisoprenoid of the first invention with an additiveto obtain a kneaded mixture; building a green tire from the kneadedmixture; and vulcanizing the green tire. With this method, a pneumatictire is produced from a polyisoprenoid produced by a method thatproduces a polyisoprenoid with high productivity. Thus, it is possibleto use plant resources effectively to produce an environmentallyfriendly pneumatic tire.

The method for producing a rubber product of the first inventionincludes the steps of: kneading a polyisoprenoid produced by the methodfor producing a polyisoprenoid of the first invention with an additiveto obtain a kneaded mixture; forming a raw rubber product from thekneaded mixture; and vulcanizing the raw rubber product. With thismethod, a rubber product is produced from a polyisoprenoid produced by amethod that produces a polyisoprenoid with high productivity. Thus, itis possible to use plant resources effectively to produce anenvironmentally friendly rubber product.

The method for producing a polyisoprenoid of the second inventionincludes producing a polyisoprenoid in a transformed plant produced byintroducing a gene coding for a cis-prenyltransferase (CPT) familyprotein, a gene coding for a Nogo-B receptor (NgBR) family protein, anda gene coding for a rubber elongation factor (REF) family protein into aplant to allow the plant to express the CPT family protein, the NgBRfamily protein, and the REF family protein. According to this method,since the CPT family protein, NgBR family protein, and REF familyprotein are co-expressed, the activity of the CPT family protein isexpected to be stabilized and enhanced. Therefore, it is expected thatthe transformed plant engineered to co-express the CPT family protein,NgBR family protein, and REF family protein exhibits continuouslyenhanced rubber synthesis activity, and the use of such a transformedplant in polyisoprenoid production can result in an increase inpolyisoprenoid production.

The method for producing a pneumatic tire of the second inventionincludes the steps of: kneading a polyisoprenoid produced by the methodfor producing a polyisoprenoid of the second invention with an additiveto obtain a kneaded mixture; building a green tire from the kneadedmixture; and vulcanizing the green tire. With this method, a pneumatictire is produced from a polyisoprenoid produced by a method thatproduces a polyisoprenoid with high productivity. Thus, it is possibleto use plant resources effectively to produce an environmentallyfriendly pneumatic tire.

The method for producing a rubber product of the second inventionincludes the steps of: kneading a polyisoprenoid produced by the methodfor producing a polyisoprenoid of the second invention with an additiveto obtain a kneaded mixture; forming a raw rubber product from thekneaded mixture; and vulcanizing the raw rubber product. With thismethod, a rubber product is produced from a polyisoprenoid produced by amethod that produces a polyisoprenoid with high productivity. Thus, itis possible to use plant resources effectively to produce anenvironmentally friendly rubber product.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a presumptive diagram of rubber synthesis by CPT, NgBR, andREF on a rubber particle.

FIG. 2 is a schematic diagram showing part of a polyisoprenoidbiosynthesis pathway.

FIG. 3 schematically illustrates the dialysis process in the example.

DESCRIPTION OF EMBODIMENTS

Herein, the first invention and the second invention are also referredto collectively as the present invention. The first invention will beexplained first, and then the second invention will be explained.

(First Invention)

The method for producing a polyisoprenoid of the first inventionincludes a step of binding a protein expressed by a gene coding for acis-prenyltransferase (CPT) family protein, a protein expressed by agene coding for a Nogo-B receptor (NgBR) family protein, and a proteinexpressed by a gene coding for a rubber elongation factor (REF) familyprotein to rubber particles in vitro.

The inventors were the first to discover that the rubber synthesis ofrubber particles is activated by binding a CPT family protein, a NgBRfamily protein, and a REF family protein to rubber particles in vitro.The inventors have also discovered here for the first time that thecombination of the CPT family protein, NgBR family protein, and REFfamily protein is directly involved in rubber synthesis. It is presumedthat the CPT family protein, NgBR family protein, and REF family proteinare disposed on rubber particle for rubber synthesis as shown in FIG. 1.FIG. 1 illustrates an example of rubber synthesis in which CPT, NgBR,and REF are shown as the CPT family protein, NgBR family protein, andREF family protein, respectively, and the isopentenyl diphosphate (IPP)substrate is polymerized by CPT to synthesize natural rubber within arubber particle.

Hence, the rubber synthesis activity of rubber particles can be enhancedby binding a CPT family protein, a NgBR family protein, and a REF familyprotein to rubber particle in vitro, for example in reaction vessels(e.g. test tubes, industrial plants) as in the production method of thefirst invention. Thus, it is possible to produce rubber more efficientlyin reaction vessels (e.g. test tubes, industrial plants).

The production method of the first invention may include any other stepas long as it involves the above binding step, and each step may beperformed once or repeated multiple times.

The amounts of the CPT family protein, NgBR family protein, and REFfamily protein to be bound to the rubber particles are not particularlylimited in the first invention.

Herein, binding of a CPT family protein, a NgBR family protein, and aREF family protein to rubber particles means that, for example, all orpart of the CPT family protein, NgBR family protein, and REF familyprotein is incorporated into the rubber particles, or inserted into themembrane structure of the rubber particles. It is not limited to theseembodiments and also includes embodiments in which, for example, theproteins are localized on the surface or inside of the rubber particles.Moreover, the concept of binding to rubber particle also includesembodiments in which the CPT family protein, NgBR family protein, andREF family protein form a complex with another protein bound to therubber particles as described above, so as to be present in the form ofthe complex on the rubber particles.

The origin of the rubber particles is not particularly limited. Forexample, the rubber particles may be derived from the latex of arubber-producing plant such as Hevea brasiliensis, Taraxacum kok-saghyz,Parthenium argentatum, Sonchus oleraceus, or Ficus elastica.

The particle size of the rubber particles is also not particularlylimited. Rubber particles of a specific particle size may be sorted outand used, or a mixture of rubber particles of different particle sizesmay be used. When rubber particles of a specific particle size aresorted out and used, the rubber particles may be either small rubberparticles (SRP) with a small particle size or large rubber particles(LRP) with a large particle size.

Commonly used methods may be employed for sorting out the rubberparticles of a specific particle size, including, for example, a methodinvolving centrifugation, preferably multistage centrifugation. Aspecific method includes centrifugation at 500-1500×g, centrifugation at1700-2500×g, centrifugation at 7000-9000×g, centrifugation at15000-25000×g, and centrifugation at 40000-60000×g, carried out in thatorder. The treatment time for each centrifugation treatment ispreferably at least 20 minutes, more preferably at least 30 minutes,still more preferably at least 40 minutes, but preferably 120 minutes orless, more preferably 90 minutes or less. The treatment temperature foreach centrifugation treatment is preferably 0° C. to 10° C., morepreferably 2° C. to 8° C., particularly preferably 4° C.

In the binding step, a protein expressed by a gene coding for acis-prenyltransferase (CPT) family protein, a protein expressed by agene coding for a Nogo-B receptor (NgBR) family protein, and a proteinexpressed by a gene coding for a rubber elongation factor (REF) familyprotein are bound to rubber particles in vitro.

The origins of the gene coding for a cis-prenyltransferase (CPT) familyprotein, the gene coding for a Nogo-B receptor (NgBR) family protein,and the gene coding for a rubber elongation factor (REF) family proteinare not particularly limited, but they are each preferably derived fromplants, more preferably at least one selected from the group consistingof plants of the genera Hevea, Sonchus, Taraxacum, and Parthenium. Amongthese, they are each still more preferably derived from at least onespecies of plant selected from the group consisting of Heveabrasiliensis, Sonchus oleraceus, Parthenium argentatum, and Taraxacumkok-saghyz, particularly preferably Hevea brasiliensis. Most preferably,they are all derived from Hevea brasiliensis.

The plant is not particularly limited, and examples include Heveaspecies such as Hevea brasiliensis; Sonchus species such as Sonchusoleraceus, Sonchus asper, and Sonchus brachyotus; Solidago species suchas Solidago altissima, Solidago virgaurea subsp. asiatica, Solidagovirgaurea subsp. leipcarpa, Solidago virgaurea subsp. leipcarpa f.paludosa, Solidago virgaurea subsp. gigantea, and Solidago gigantea Ait.var. leiophylla Fernald; Helianthus species such as Helianthus annus,Helianthus argophyllus, Helianthus atrorubens, Helianthus debilis,Helianthus decapetalus, and Helianthus giganteus; Taraxacum species suchas dandelion (Taraxacum), Taraxacum venustum H. Koidz, Taraxacumhondoense Nakai, Taraxacum platycarpum Dahlst, Taraxacum japonicum,Taraxacum officinale Weber, and Taraxacum kok-saghyz; Ficus species suchas Ficus carica, Ficus elastica, Ficus pumila L., Ficus erecta Thumb.,Ficus ampelas Burm. f., Ficus benguetensis Merr., Ficus irisana Elm.,Ficus microcarpa L. f., Ficus septica Burm. f., and Ficus benghalensis;Parthenium species such as Parthenium argentatum, and Partheniumhysterophorus, Ambrosia artemisiifolia; and lettuce (Lactuca sativa).

Herein, the cis-prenyltransferase (CPT) family protein refers to anenzyme that catalyzes a reaction of cis-chain elongation of anisoprenoid compound. Specifically, in plants, for example,polyisoprenoids are biosynthesized via polyisoprenoid biosynthesispathways as shown in FIG. 2, in which the CPT family proteins areconsidered to be enzymes that catalyze the reaction enclosed by thedotted frame in FIG. 2. The CPT family proteins are characterized byhaving an amino acid sequence contained in the cis-IPPS domain (NCBIaccession No. cd00475). Examples of such CPT family proteins include CPTfrom Hevea brasiliensis (HRT1, HRT2, CPT3-5), AtCPT1-9 from Arabidopsisthaliana, CPT1-3 from lettuce, CPT1-3 from Taraxacum kok-saghyz, andundecaprenyl pyrophosphate synthase (UPPS) from E. coli.

Herein, the isoprenoid compound refers to a compound containing anisoprene unit (C₅H₈). Also, the cis isoprenoid refers to a compoundincluding an isoprenoid compound in which isoprene units are cis-bonded,and examples include cis-farnesyl diphosphate, undecaprenyl diphosphate,natural rubber, and the like.

The Nogo-B receptor (NgBR) family protein refers to a protein having thefunctions of binding to a membrane via one or more transmembrane domainson the N-terminal side of the protein, and interacting with CPT familyproteins or other proteins on the C-terminal side thereof, and assiststhe function of the CPT family proteins by holding the CPT familyproteins on the membrane. The NgBR family proteins are characterized byhaving a transmembrane domain on the N-terminal side, and further havingan amino acid sequence contained in the cis-IPPS superfamily domain(NCBI accession No. COG0020) on the C-terminal side. Examples of suchNgBR family proteins include NgBR from Hevea brasiliensis (HRTBP), LEW1from Arabidopsis thaliana, LsCPTL1-2 from lettuce, and TbRTA fromTaraxacum.

The rubber elongation factor (REF) family protein refers to a rubberparticle-associated protein that is bound to rubber particles in thelatex of rubber-producing plants such as Hevea brasiliensis, andcontributes to stabilization of the rubber particles. The REF familyproteins are characterized by having an amino acid sequence contained inthe REF superfamily domain (NCBI accession No. pfam05755). Examples ofsuch REF family proteins include REF, small rubber particle protein(SRPP) and the like.

Specific examples of the CPT family protein include the following [1]:

[1] a protein having the amino acid sequence of SEQ ID NO:2.

It is known that proteins having one or more amino acid substitutions,deletions, insertions, or additions relative to the original amino acidsequence can have the inherent function. Thus, specific examples of theCPT family protein also include the following [2]:

[2] a protein having an amino acid sequence containing one or more aminoacid substitutions, deletions, insertions and/or additions relative tothe amino acid sequence of SEQ ID NO:2, and having an enzyme activitythat catalyzes a reaction of cis-chain elongation of an isoprenoidcompound.

In order to preserve the function of the CPT family protein, itpreferably has an amino acid sequence containing one or more, morepreferably 1-58, still more preferably 1-44, further more preferably1-29, particularly preferably 1-15, most preferably 1-6, yet mostpreferably 1-3 amino acid substitutions, deletions, insertions and/oradditions relative to the amino acid sequence of SEQ ID NO:2.

Among other amino acid substitutions, conservative substitutions arepreferred. Specific examples include substitutions within each of thefollowing groups in the parentheses: (glycine, alanine), (valine,isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine,glutamine), (serine, threonine), (lysine, arginine), (phenylalanine,tyrosine), and the like.

It is also known that proteins with amino acid sequences having highsequence identity with the original amino acid sequence can also havesimilar function. Thus, specific examples of the CPT family protein alsoinclude the following [3]:

[3] a protein having an amino acid sequence having at least 80% sequenceidentity with the amino acid sequence of SEQ ID NO:2, and having anenzyme activity that catalyzes a reaction of cis-chain elongation of anisoprenoid compound.

In order to preserve the function of the CPT family protein, thesequence identity with the amino acid sequence of SEQ ID NO:2 ispreferably at least 85%, more preferably at least 90%, still morepreferably at least 95%, particularly preferably at least 98%, mostpreferably at least 99%.

The sequence identity between amino acid sequences or nucleotidesequences may be determined using the algorithm BLAST [Pro. Natl. Acad.Sci. USA, 90, 5873 (1993)] developed by Karlin and Altschul or FASTA[Methods Enzymol., 183, 63 (1990)] (all the above documents areincorporated herein by reference).

Whether it is a protein having the above enzyme activity may bedetermined by conventional techniques, such as by expressing a targetprotein in a transformant prepared by introducing a gene coding for thetarget protein into Escherichia coli or other host organisms, anddetermining the presence or absence of the function of the targetprotein by the corresponding activity measurement method.

Specific examples of the NgBR family protein include the following [4]:

[4] a protein having the amino acid sequence of SEQ ID NO:4.

It is known that proteins having one or more amino acid substitutions,deletions, insertions, or additions relative to the original amino acidsequence can have the inherent function. Thus, specific examples of theNgBR family protein also include the following [5]:

[5] a protein having an amino acid sequence containing one or more aminoacid substitutions, deletions, insertions and/or additions relative tothe amino acid sequence of SEQ ID NO:4, and having the functions ofbinding to a membrane via one or more transmembrane domains on theN-terminal side of the protein, and interacting with another protein onthe C-terminal side thereof.

In order to preserve the function of the NgBR family protein, itpreferably has an amino acid sequence containing one or more, morepreferably 1-52, still more preferably 1-39, further more preferably1-26, particularly preferably 1-13, most preferably 1-6, yet mostpreferably 1-3 amino acid substitutions, deletions, insertions and/oradditions relative to the amino acid sequence of SEQ ID NO:4.

Among other amino acid substitutions, conservative substitutions arepreferred. Specific examples include substitutions within each of thefollowing groups in the parentheses: (glycine, alanine), (valine,isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine,glutamine), (serine, threonine), (lysine, arginine), (phenylalanine,tyrosine), and the like.

As described above, it is also known that proteins with amino acidsequences having high sequence identity with the original amino acidsequence can also have similar function. Thus, specific examples of theNgBR family protein also include the following [6]:

[6] a protein having an amino acid sequence having at least 80% sequenceidentity with the amino acid sequence of SEQ ID NO:4, and having thefunctions of binding to a membrane via one or more transmembrane domainson the N-terminal side of the protein, and interacting with anotherprotein on the C-terminal side thereof.

In order to preserve the function of the NgBR family protein, thesequence identity with the amino acid sequence of SEQ ID NO:4 ispreferably at least 85%, more preferably at least 90%, still morepreferably at least 95%, particularly preferably at least 98%, mostpreferably at least 99%.

Whether it is a NgBR family protein may be determined by conventionaltechniques, such as by identifying the amino acid sequence and thendetermining whether it has an amino acid sequence contained in thecis-IPPS superfamily domain (NCBI accession No. COG0020).

Specific examples of the REF family protein include the following [7]:

[7] a protein having the amino acid sequence of SEQ ID NO:6.

It is known that proteins having one or more amino acid substitutions,deletions, insertions, or additions relative to the original amino acidsequence can have the inherent function. Thus, specific examples of theREF family protein also include the following [8]:

[8] a rubber particle-associated protein having an amino acid sequencecontaining one or more amino acid substitutions, deletions, insertions,and/or additions relative to the amino acid sequence of SEQ ID NO:6, andbeing bound to rubber particles in latex.

In order to preserve the function of the REF family protein, itpreferably has an amino acid sequence containing one more, morepreferably 1-28, still more preferably 1-21, further more preferably1-14, particularly preferably 1-7, most preferably 1-3, yet mostpreferably one amino acid substitution, deletion, insertion and/oraddition relative to the amino acid sequence of SEQ ID NO:6.

Among other amino acid substitutions, conservative substitutions arepreferred. Specific examples include substitutions within each of thefollowing groups in the parentheses: (glycine, alanine), (valine,isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine,glutamine), (serine, threonine), (lysine, arginine), (phenylalanine,tyrosine), and the like.

As described above, it is also known that proteins with amino acidsequences having high sequence identity with the original amino acidsequence can also have similar function. Thus, specific examples of theREF family protein also include the following [9]:

[9] a rubber particle-associated protein having an amino acid sequencehaving at least 80% sequence identity with the amino acid sequence ofSEQ ID NO:6, and being bound to rubber particles in latex.

In order to preserve the function of the REF family protein, thesequence identity with the amino acid sequence of SEQ ID NO:6 ispreferably at least 85%, more preferably at least 90%, still morepreferably at least 95%, particularly preferably at least 98%, mostpreferably at least 99%.

Whether it is a REF family protein may be determined by conventionaltechniques, such as by identifying the amino acid sequence and thendetermining whether it has an amino acid sequence contained in the REFsuperfamily domain (NCBI accession No. pfam05755).

Specific examples of the gene coding for the CPT family protein includethe following [1] and [2]:

[1] a DNA having the nucleotide sequence of SEQ ID NO:1; and

[2] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:1, and codes for a protein having an enzyme activity that catalyzes areaction of cis-chain elongation of an isoprenoid compound.

As used herein, the term “hybridizing” means a process in which a DNAhybridizes with a DNA having a specific nucleotide sequence or a part ofthe DNA. Accordingly, the DNA having a specific nucleotide sequence orpart of the DNA may have a nucleotide sequence long enough to be usableas a probe in Northern or Southern blot analysis or as anoligonucleotide primer in polymerase chain reaction (PCR) analysis. TheDNA used as a probe may have a length of at least 100 bases, preferablyat least 200 bases, more preferably at least 500 bases although it maybe a DNA of at least 10 bases, preferably of at least bases in length.

Techniques to perform DNA hybridization experiments are well known. Thehybridization conditions under which experiments are carried out may bedetermined according to, for example, Molecular Cloning, 2nd ed. and 3rded. (2001), Methods for General and Molecular Bacteriology, ASM Press(1994), Immunology methods manual, Academic press (Molecular), and manyother standard textbooks (all the above documents are incorporatedherein by reference).

The stringent conditions may include, for example, an overnightincubation at 42° C. of a DNA-immobilized filter and a DNA probe in asolution containing 50% formamide, 5×SSC (750 mM sodium chloride, 75 mMsodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution,10% dextran sulfate, and 20 μg/l denatured salmon sperm DNA, followed bywashing the filter for example in a 0.2×SSC solution at approximately65° C. Less stringent conditions may also be used. Changes in thestringency may be accomplished through the manipulation of formamideconcentration (lower percentages of formamide result in lowerstringency), salt concentrations or temperature. For example, lowstringent conditions include an overnight incubation at 37° C. in asolution containing 6×SSCE (20×SSCE: 3 mol/l sodium chloride, 0.2 mol/lsodium dihydrogen phosphate, 0.02 mol/l EDTA, pH 7.4), 0.5% SDS, 30%formamide, and 100 μg/l denatured salmon sperm DNA, followed by washingin a ix SSC solution containing 0.1% SDS at 50° C. In addition, toachieve even lower stringency, washes performed following hybridizationmay be done at higher salt concentrations (e.g. 5×SSC) in theabove-mentioned low stringent conditions.

Variations in the above various conditions may be accomplished throughthe inclusion or substitution of blocking reagents used to suppressbackground in hybridization experiments. The inclusion of blockingreagents may require modification of the hybridization conditions forcompatibility.

The DNA capable of hybridizing under such stringent conditions may havea nucleotide sequence having at least 80%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%,particularly preferably at least 99% sequence identity with thenucleotide sequence of SEQ ID NO: 1 as calculated using a program suchas BLAST or FASTA with the above-mentioned parameters.

Whether the DNA that hybridizes under stringent conditions with theaforementioned DNA codes for a protein having a predetermined enzymeactivity may be determined by conventional techniques, such as byexpressing a target protein in a transformant prepared by introducing agene coding for the target protein into Escherichia coli or other hostorganisms, and determining the presence or absence of the function ofthe target protein by the corresponding activity measurement method.

Specific examples of the gene coding for the NgBR family protein includethe following [3] and [4]:

[3] a DNA having the nucleotide sequence of SEQ ID NO:3; and

[4] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:3, and codes for a protein having the functions of binding to amembrane via one or more transmembrane domains on the N-terminal side ofthe protein, and interacting with another protein on the C-terminal sidethereof.

The term “hybridizing” is as described above. Also, the stringentconditions are as described above.

The DNA capable of hybridizing under such stringent conditions may havea nucleotide sequence having at least 80%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%,particularly preferably at least 99% sequence identity with thenucleotide sequence of SEQ ID NO: 3 as calculated using a program suchas BLAST or FASTA with the above-mentioned parameters.

Whether the DNA that hybridizes under stringent conditions with theaforementioned DNA codes for a NgBR family protein may be determined byconventional techniques, such as by translating the DNA into an aminoacid sequence and then determining whether the amino acid sequence hasan amino acid sequence contained in the cis-IPPS superfamily domain(NCBI accession No. COG0020).

Specific examples of the gene coding for the REF family protein includethe following [5] and [6]:

[5] a DNA having the nucleotide sequence of SEQ ID NO:5; and

[6] a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:5, and codes for a rubber particle-associated protein that is boundto rubber particles in latex.

The term “hybridizing” is as described above. Also, the stringentconditions are as described above.

The DNA capable of hybridizing under such stringent conditions may havea nucleotide sequence having at least 80%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 98%,particularly preferably at least 99% sequence identity with thenucleotide sequence of SEQ ID NO: 5 as calculated using a program suchas BLAST or FASTA with the above-mentioned parameters.

Whether the DNA that hybridizes under stringent conditions with theaforementioned DNA codes for a REF family protein may be determined byconventional techniques, such as by translating the DNA into an aminoacid sequence and then determining whether the amino acid sequence hasan amino acid sequence contained in the REF superfamily domain (NCBIaccession No. pfam05755).

Also, conventional techniques may be employed to identify the amino acidsequence or the nucleotide sequence of the proteins. For example, totalRNA is extracted from a growing plant, the mRNA is optionally purified,and a cDNA is synthesized by a reverse transcription reaction.Subsequently, degenerate primers are designed based on the amino acidsequence of a known protein corresponding to the target protein, a DNAfragment is partially amplified by RT-PCR, and the sequence is partiallyidentified. Then, the RACE method or the like is performed to identifythe full-length nucleotide sequence or amino acid sequence. The RACEmethod (rapid amplification of cDNA ends method) refers to a method inwhich, when the nucleotide sequence of a cDNA is partially known, PCR isperformed based on the nucleotide sequence information of such a knownregion to clone an unknown region extending to the cDNA terminal, and iscapable of cloning the full-length cDNA by PCR without preparing a cDNAlibrary.

The degenerate primers may each preferably be prepared from aplant-derived sequence having a highly similar sequence part to thetarget protein.

If the nucleotide sequence coding for the protein is known, it ispossible to identify the full-length nucleotide sequence or amino acidsequence by designing a primer containing an initiation codon and aprimer containing a termination codon using the known nucleotidesequence followed by performing RT-PCR using a synthesized cDNA as atemplate.

In the binding step, another protein may further be bound to the rubberparticles as long as the protein expressed by a gene coding for acis-prenyltransferase (CPT) family protein, the protein expressed by agene coding for a Nogo-B receptor (NgBR) family protein, and the proteinexpressed by a gene coding for a rubber elongation factor (REF) familyprotein are bound to the rubber particles in vitro.

The origin of the other protein is not particularly limited, butpreferably it is derived from any of the plants described above, morepreferably at least one selected from the group consisting of plants ofthe genera Hevea, Sonchus, Taraxacum, and Parthenium. Among these, it isstill more preferably derived from at least one species of plantselected from the group consisting of Hevea brasiliensis, Sonchusoleraceus, Parthenium argentatum, and Taraxacum kok-saghyz, particularlypreferably Hevea brasiliensis.

The other protein may be any protein without any limitations, but forpurposes of enhancing the rubber synthesis activity of the rubberparticles, it is preferably a protein that inherently exists on rubberparticles in rubber-producing plants. The protein that exists on rubberparticles may be a protein that binds to a large part of the membranesurface of rubber particles, or a protein that is inserted into andbound to the membrane of rubber particles, or a protein that forms acomplex with another protein bound to the membrane so as to be presenton the membrane surface.

Examples of the protein that inherently exists on rubber particles inrubber-producing plants include β-1,3-glucanase, and Hevein.

The binding step may be carried out by any method that binds the CPTfamily protein, NgBR family protein, and REF family protein to rubberparticles in vitro, such as, for example, by performing proteinsynthesis in the presence of both rubber particles and a cell-freeprotein synthesis solution containing an mRNA coding for the CPT familyprotein, an mRNA coding for the NgBR family protein, and an mRNA codingfor the REF family protein to bind the CPT family protein, the NgBRfamily protein, and the REF family protein to the rubber particles.

The binding step preferably includes performing protein synthesis in thepresence of both rubber particles and a cell-free protein synthesissolution containing an mRNA coding for a CPT family protein, an mRNAcoding for a NgBR family protein, and an mRNA coding for a REF familyprotein to bind the CPT family protein, the NgBR family protein, and theREF family protein to the rubber particles, among other methods.

In other words, rubber particles bound to a CPT family protein, a NgBRfamily protein, and a REF family protein are preferably obtained byperforming protein synthesis in the presence of both rubber particlesand a cell-free protein synthesis solution containing mRNAs coding forthe CPT family protein, the NgBR family protein, and the REF familyprotein, or, more specifically, using a mixture of rubber particles witha cell-free protein synthesis solution containing mRNAs coding for theCPT family protein, the NgBR family protein, and the REF family protein.

Since liposomes are produced artificially as lipid bilayer membranesconsisting of phospholipids, glyceroglycolipids, cholesterol and othercomponents, the produced liposomes have no proteins bound to theirsurface. On the other hand, although rubber particles harvested from thelatex of rubber-producing plants are also coated with a lipid membrane,the membrane of rubber particles is a naturally derived membrane inwhich proteins that have been synthesized in the plants are alreadybound to the surface of the membrane. Hence, binding of an additionalprotein to rubber particles that are already bound to and coated withproteins is expected to be more difficult than binding to liposomes notbound to any protein. There is also concern that the proteins alreadybound to rubber particles could inhibit cell-free protein synthesis. Forthese reasons, difficulties have been anticipated in achieving cell-freeprotein synthesis in the presence of rubber particles. Under suchcircumstances, the present inventors have first discovered that rubberparticles bound to a CPT family protein, a NgBR family protein, and aREF family protein can be produced by performing cell-free synthesis ofthe CPT family protein, the NgBR family protein, and the REF familyprotein in the presence of rubber particles, which had never beenattempted in the past.

The protein synthesis in the presence of both rubber particles and acell-free protein synthesis solution containing mRNAs coding for a CPTfamily protein, a NgBR family protein, and a REF family protein isnamely the synthesis of a CPT family protein, a NgBR family protein, anda REF family protein by cell-free protein synthesis, and the synthesizedCPT family protein, NgBR family protein, and REF family protein maintainbiological functions (the native state). As the cell-free proteinsynthesis is performed in the presence of rubber particles, thesynthesized CPT family protein, NgBR family protein, and REF familyprotein in the native state can be bound to the rubber particles.

Herein, binding of a CPT family protein, a NgBR family protein, and aREF family protein to rubber particles by protein synthesis in thepresence of both the cell-free protein synthesis solution and the rubberparticles means that, for example, all or part of the CPT familyprotein, NgBR family protein, and REF family protein synthesized by theprotein synthesis is incorporated into the rubber particles, or insertedinto the membrane structure of the rubber particles. It is not limitedto these embodiments and also includes embodiments in which, forexample, the proteins are localized on the surface or inside of therubber particles. Moreover, the concept of binding to rubber particlealso includes embodiments in which the proteins form a complex withanother protein bound to the rubber particles as described above, so asto be present in the form of the complex on the rubber particles.

The mRNAs coding for a CPT family protein, a NgBR family protein, and aREF family protein serve as translation templates that can be translatedto synthesize the CPT family protein, NgBR family protein, and REFfamily protein, respectively.

The origins of the mRNAs coding for a CPT family protein, a NgBR familyprotein, and a REF family protein are not particularly limited, butpreferably they are each derived from any of the plants described above,more preferably at least one selected from the group consisting ofplants of the genera Hevea, Sonchus, Taraxacum, and Parthenium. Amongthese, they are each still more preferably derived from at least onespecies of plant selected from the group consisting of Heveabrasiliensis, Sonchus oleraceus, Parthenium argentatum, and Taraxacumkok-saghyz, particularly preferably Hevea brasiliensis.

The methods for preparing the mRNAs coding for a CPT family protein, aNgBR family protein, and a REF family protein are not particularlylimited as long as the prepared mRNAs serve as translation templatesthat can be translated to synthesize the CPT family protein, NgBR familyprotein, and REF family protein. For example, the mRNAs may be preparedby extracting total RNA from the latex of a rubber-producing plant by,for example, the hot phenol method, synthesizing cDNA from the totalRNA, obtaining a DNA fragment of a gene coding for a CPT family protein,NgBR family protein, or REF family protein using primers prepared basedon the nucleotide sequence data of the gene coding for a CPT familyprotein, NgBR family protein, or REF family protein, and performing anordinary in vitro transcription of the DNA fragment.

As long as the cell-free protein synthesis solution contains the mRNAscoding for a CPT family protein, a NgBR family protein, and a REF familyprotein, it may contain a mRNA coding for another protein.

The mRNA coding for another protein may be an mRNA that can betranslated to express the other protein. The other protein may be asdescribed above.

In the binding step in the first invention, cell-free synthesis of a CPTfamily protein, a NgBR family protein, and a REF family protein ispreferably performed in the presence of rubber particles. This cell-freeprotein synthesis may be carried out by methods similar to conventionalmethods using the cell-free protein synthesis solution. Commonly usedcell-free protein synthesis techniques may be employed for the cell-freeprotein synthesis system, such as a rapid translation system RTS500(Roche Diagnostics); and wheat germ extracts prepared in accordance withProc. Natl. Acad. Sci. USA, 97:559-564 (2000), JP-A 2000-236896, JP-A2002-125693 or JP-A 2002-204689, and cell-free protein synthesis systemsusing the wheat germ extracts (JP-A 2002-204689, Proc. Natl. Acad. Sci.USA, 99:14652-14657 (2002)). All the above documents are incorporatedherein by reference. Systems using germ extracts are preferred amongthese. Thus, in another suitable embodiment of the first invention, thecell-free protein synthesis solution contains a germ extract.

The source of the germ extract is not particularly limited. From thestandpoint of translation efficiency, it is preferred to use aplant-derived germ extract when a plant protein is synthesized bycell-free protein synthesis. It is particularly preferred to use awheat-derived germ extract. Thus, in another suitable embodiment of thefirst invention, the germ extract is derived from wheat.

The method for preparing the germ extract is not particularly limited,and may be carried out conventionally, as described in, for example,JP-A 2005-218357, incorporated herein by reference.

The cell-free protein synthesis solution preferably further contains acyclic nucleoside monophosphate derivative or a salt thereof(hereinafter, also referred to simply as “activity enhancer”). Proteinsynthesis activity can be further enhanced by the inclusion of theactivity enhancer.

The cyclic nucleoside monophosphate derivative or salt thereof is notparticularly limited as long as it can enhance cell-free proteinsynthesis activity, and examples include adenosine-3′,5′-cyclicmonophosphoric acid and its salts; adenosine-3′,5′-cyclicmonophosphorothioic acid (Sp-isomer) and its salts;adenosine-3′,5′-cyclic monophosphorothioic acid (Rp-isomer) and itssalts; guanosine-3′,5′-cyclic monophosphoric acid and its salts;guanosine-3′,5′-cyclic monophosphorothioic acid (Sp-isomer) and itssalts; guanosine-3′,5′-cyclic monophosphorothioic acid (Rp-isomer) andits salts; 8-bromoadenosine-3′,5′-cyclic monophosphoric acid(bromo-cAMP) and its salts; 8-(4-chlorophenylthio)adenosine-3′,5′-cyclicmonophosphoric acid (chlorophenylthio-cAMP) and its salts;5,6-dichloro-1-β-D-ribofuranosylbenzimidazole adenosine-3′,5′-cyclicmonophosphoric acid (dichlororibofuranosylbenzimidazole cAMP) and itssalts; adenosine-2′,5′-cyclic monophosphoric acid and its salts;adenosine-2′,5′-cyclic monophosphorothioic acid (Sp-isomer) and itssalts; adenosine-2′,5′-cyclic monophosphorothioic acid (Rp-isomer) andits salts; guanosine-2′,5′-cyclic monophosphoric acid and its salts;guanosine-2′,5′-cyclic monophosphorothioic acid (Sp-isomer) and itssalts; and guanosine-2′,5′-cyclic monophosphorothioic acid (Rp-isomer)and its salts.

The base that forms a salt with the cyclic nucleoside monophosphatederivative is not particularly limited as long as it is biochemicallyacceptable and forms a salt with the derivative. Preferred are, forexample, alkali metal atoms such as sodium or potassium, and organicbases such as Tris-hydroxyaminomethane, among others.

Of these activity enhancers, adenosine-3′,5′-cyclic monophosphoric acidor adenosine-3′,5′-cyclic monophosphate sodium is particularlypreferred. These activity enhancers may be used alone or in combinationsof two or more.

The activity enhancer may be incorporated into the cell-free proteinsynthesis solution in advance. If the activity enhancer is unstable inthe solution, it is preferably added during the protein synthesisreaction performed in the presence of both the cell-free proteinsynthesis solution and rubber particles.

The amount of the activity enhancer added is not particularly limited aslong as the activity enhancer is at a concentration that can activate(increase) the protein synthesis reaction in the cell-free proteinsynthesis solution. Specifically, the final concentration in thereaction system may usually be at least 0.1 millimoles/liter. The lowerlimit of the concentration is preferably 0.2 millimoles/liter, morepreferably 0.4 millimoles/liter, particularly preferably 0.8millimoles/liter, while the upper limit of the concentration ispreferably 24 millimoles/liter, more preferably 6.4 millimoles/liter,particularly preferably 3.2 millimoles/liter.

When adding the activity enhancer to the cell-free protein synthesissolution, the temperature of the cell-free protein synthesis solution isnot particularly limited, but is preferably 0° C. to 30° C., morepreferably 10° C. to 26° C.

In addition to the mRNAs (translation templates) coding for a CPT familyprotein, a NgBR family protein, and a REF family protein, the cell-freeprotein synthesis solution also contains ATP, GTP, creatine phosphate,creatine kinase, L-amino acids, potassium ions, magnesium ions and othercomponents required for protein synthesis, and optionally an activityenhancer. Such a cell-free protein synthesis solution can serve as acell-free protein synthesis reaction system.

Since the germ extract prepared by the method described in JP-A2005-218357 contains tRNA in an amount necessary for protein synthesisreaction, addition of separately prepared tRNA is not required when thegerm extract prepared by the above method is used in the cell-freeprotein synthesis solution. In other words, tRNA may be added to thecell-free protein synthesis solution as necessary.

The binding step in the first invention preferably includes performingprotein synthesis in the presence of both rubber particles and acell-free protein synthesis solution containing mRNAs coding for a CPTfamily protein, a NgBR family protein, and a REF family protein.Specifically, this can be accomplished by adding rubber particles to thecell-free protein synthesis solution at a suitable point either beforeor after protein synthesis, preferably before protein synthesis.

The rubber particles are preferably present in the cell-free proteinsynthesis solution at a concentration of 5 to 50 g/L. In other words, 5to 50 g of rubber particles are preferably present in 1 L of thecell-free protein synthesis solution. When the concentration of rubberparticles present in the cell-free protein synthesis solution is lessthan 5 g/L, a rubber layer may not be formed by separation treatment(e.g. ultracentrifugation) for collecting the rubber particles bound tothe synthesized CPT family protein, NgBR family protein, and REF familyprotein, and therefore it may be difficult to collect the rubberparticles bound to the synthesized CPT family protein, NgBR familyprotein, and REF family protein. When the concentration of rubberparticles present in the cell-free protein synthesis solution exceeds 50g/L, the rubber particles may coagulate, so that the synthesized CPTfamily protein, NgBR family protein, and REF family protein may fail tobind well to the rubber particles. The concentration of rubber particlesis more preferably 10 to 40 g/L, still more preferably 15 to 35 g/L,particularly preferably 15 to 30 g/L.

In the protein synthesis in the presence of both rubber particles andthe cell-free protein synthesis solution, rubber particles may be addedas appropriate as the reaction progresses. The cell-free proteinsynthesis solution and rubber particles are preferably present togetherduring the period when the cell-free protein synthesis system is active,such as 3 to 48 hours, preferably 3 to 30 hours, more preferably 3 to 24hours after the addition of rubber particles to the cell-free proteinsynthesis solution.

The rubber particles do not have to be subjected to any treatment, e.g.pretreatment, before use in the binding step in the first invention,preferably before being combined with the cell-free protein synthesissolution. However, proteins may be removed from the rubber particleswith a surfactant beforehand to increase the proportions of the CPTfamily protein, NgBR family protein, and REF family protein desired tobe bound by the method of the first invention, among the proteinspresent on the rubber particles. Thus, in another suitable embodiment ofthe first invention, the rubber particles used in the first inventionare washed with a surfactant before use in the binding step in the firstinvention, preferably before being combined with the cell-free proteinsynthesis solution.

The surfactant is not particularly limited, and examples includenonionic surfactants and amphoteric surfactants. Nonionic surfactantsand amphoteric surfactants, among others, are suitable because they haveonly a little denaturing effect on the proteins on the membrane, andamphoteric surfactants are especially suitable. Thus, in anothersuitable embodiment of the first invention, the surfactant is anamphoteric surfactant.

These surfactants may be used alone or in combinations of two or more.

Examples of nonionic surfactants include polyoxyalkylene ether nonionicsurfactants, polyoxyalkylene ester nonionic surfactants, polyvalentalcohol fatty acid ester nonionic surfactants, sugar fatty acid esternonionic surfactants, alkyl polyglycoside nonionic surfactants, andpolyoxyalkylene polyglucoside nonionic surfactants; and polyoxyalkylenealkylamines and alkyl alkanolamides.

Of these, polyoxyalkylene ether nonionic surfactants or polyvalentalcohol fatty acid ester nonionic surfactants are preferred.

Examples of polyoxyalkylene ether nonionic surfactants includepolyoxyalkylene alkyl ethers, polyoxyalkylene alkylphenyl ethers,polyoxyalkylene polyol alkyl ethers, and polyoxyalkylene mono-, di- ortristyryl phenyl ethers. Among these, polyoxyalkylene alkylphenyl ethersare suitable. The polyol is preferably a C₂₋₁₂ polyvalent alcohol, suchas ethylene glycol, propylene glycol, glycerin, sorbitol, glucose,sucrose, pentaerythritol, or sorbitan.

Examples of polyoxyalkylene ester nonionic surfactants includepolyoxyalkylene fatty acid esters and polyoxyalkylene alkyl rosin acidesters.

Examples of polyvalent alcohol fatty acid ester nonionic surfactantsinclude fatty acid esters of C₂₋₁₂ polyvalent alcohols and fatty acidesters of polyoxyalkylene polyvalent alcohols. More specific examplesinclude sorbitol fatty acid esters, sorbitan fatty acid esters, glycerinfatty acid esters, polyglycerin fatty acid esters, and pentaerythritolfatty acid esters, as well as polyalkylene oxide adducts of theforegoing (e.g. polyoxyalkylene sorbitan fatty acid esters,polyoxyalkylene glycerin fatty acid esters). Among these, sorbitan fattyacid esters are suitable.

Examples of sugar fatty acid ester nonionic surfactants include fattyacid esters of sucrose, glucose, maltose, fructose and polysaccharides,as well as polyalkylene oxide adducts of the foregoing.

Examples of alkyl polyglycoside nonionic surfactants include thosehaving glucose, maltose, fructose, sucrose and the like as glycosides,such as alkyl glucosides, alkyl polyglucosides, polyoxyalkylene alkylglucosides, and polyoxyalkylene alkyl polyglucosides, as well as fattyacid esters of the foregoing. Polyalkylene oxide adducts of any of theforegoing may also be used.

Examples of the alkyl groups in these nonionic surfactants include C₄₋₃₀linear or branched, saturated or unsaturated alkyl groups. Thepolyoxyalkylene groups may have C₂₋₄ alkylene groups, and may have about1-50 moles of added ethylene oxide, for example. Examples of the fattyacids include C₄₋₃₀ linear or branched, saturated or unsaturated fattyacids.

Of the nonionic surfactants, polyoxyethylene (10) octylphenyl ether(Triton X-100) or sorbitan monolaurate (Span 20) is particularlypreferred for their ability to moderately remove membrane-bound proteinswhile keeping the membrane of rubber particle stable and, further,having only a little denaturing effect on the proteins.

Examples of amphoteric surfactants include zwitterionic surfactants suchas quaternary ammonium group/sulfonate group (—SO₃H) surfactants,water-soluble quaternary ammonium group/phosphate group surfactants,water-insoluble quaternary ammonium group/phosphate group surfactants,and quaternary ammonium group/carboxyl group surfactants. The acidgroups in these zwitterionic surfactants may be salts.

In particular, the zwitterionic surfactant preferably has both positiveand negative charges in a molecule, and the acid dissociation constant(pKa) of the acid group is preferably 5 or less, more preferably 4 orless, still more preferably 3 or less.

Specific examples of the amphoteric surfactant include ammoniumsulfobetaines such as3-[3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propanesulfonate(CHAPSO), 3-[3-cholamidopropyl)-dimethylamino]-propanesulfonate (CHAPS),N,N-bis(3-D-gluconamidopropyl)-cholamide,n-octadecyl-N,N′-dimethyl-3-amino-1-propanesulfonate,n-decyl-N,N′-dimethyl-3-amino-1-propanesulfonate,n-dodecyl-N,N′-dimethyl-3-amino-1-propanesulfonate,n-tetradecyl-N,N′-dimethyl-3-amino-1-propanesulfonate(Zwittergent™-3-14},n-hexadecyl-N,N′-dimethyl-3-amino-1-propanesulfonate, andn-octadecyl-N,N′-dimethyl-3-amino-1-propanesulfonate; phosphocholinessuch as n-octylphosphocholine, n-nonylphosphocholine,n-decylphosphocholine, n-dodecylphosphocholine,n-tetradecylphosphocholine, and n-hexadecylphosphocholine; andphosphatidylcholines such as dilauroyl phosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoylphosphatidylcholine, dioleoyl phosphatidylcholine, and dilinoleoylphosphatidylcholine. Of these,3-[(3-cholamidopropyl)dimethylamino]-propanesulfonate (CHAPS) isparticularly preferred for its ability to moderately remove proteinswhile keeping the membrane of rubber particles stable.

The concentration of the surfactant for the treatment is preferablywithin three times the critical micelle concentration (CMC) of thesurfactant used. The membrane stability of the rubber particles may bereduced if they are treated with the surfactant at a concentrationexceeding three times the critical micelle concentration. Theconcentration is more preferably within 2.5 times, still more preferablywithin 2.0 times the CMC. The lower limit of the concentration ispreferably 0.05 times or more, more preferably 0.1 times or more, stillmore preferably 0.3 times or more the CMC.

Examples of reaction systems or apparatuses that can be used in thecell-free protein synthesis include a batch method (Pratt, J. M. et al.,Transcription and Translation, Hames, 179-209, B. D. & Higgins, S. J.,eds, IRL Press, Oxford (1984)), a continuous cell-free protein synthesissystem in which amino acids, energy sources and the like are suppliedcontinuously to the reaction system (Spirin, A. S. et al., Science, 242,1162-1164 (1988)), a dialysis method (Kigawa et al., 21st Annual Meetingof the Molecular Biology Society of Japan, WID 6) and an overlay method(instruction manual of PROTEIOS™ wheat germ cell-free protein synthesiscore kit, Toyobo Co., Ltd.). All the above documents are incorporatedherein by reference. Another method may be to supply template RNA, aminoacids, energy sources and the like as necessary to the protein synthesisreaction system, and discharge the synthesis product or decompositionproduct as required.

Among these, the overlay method has the advantage of easy operation, butunfortunately the rubber particles disperse in the reaction solution andthus are difficult to efficiently bind to the synthesized CPT familyprotein, NgBR family protein, and REF family protein, while, in thedialysis method, since the amino acids used as raw materials of the CPTfamily protein, NgBR family protein, and REF family protein to besynthesized can pass through the dialysis membrane but the rubberparticles cannot pass therethrough, the dispersal of the rubberparticles can be prevented, and thus it is possible to efficiently bindthe synthesized CPT family protein, NgBR family protein, and REF familyprotein to the rubber particles. Accordingly, the dialysis method ispreferred.

The dialysis method refers to a method in which protein synthesis iscarried out using the reaction solution for the cell-free proteinsynthesis as an inner dialysis solution, and an apparatus in which theinner dialysis solution is separated from an outer dialysis solution bya dialysis membrane capable of mass transfer. Specifically, for example,a translation template is added to the synthesis reaction solutionexcluding the translation template, optionally after pre-incubation foran appropriate amount of time, and then the solution is put in asuitable dialysis container as the inner reaction solution. Examples ofthe dialysis container include containers with a dialysis membraneattached to the bottom (e.g. Dialysis Cup 12000 available from DaiichiKagaku) and dialysis tubes (e.g. 12000 available from Sanko Junyaku Co.,Ltd.). The dialysis membrane used has a molecular weight cutoff of10,000 daltons or more, preferably about 12,000 daltons.

The outer dialysis solution used is a buffer containing amino acids. Thedialysis efficiency can be increased by replacing the outer dialysissolution with a fresh solution when the reaction speed declines. Thereaction temperature and time are selected appropriately according tothe protein synthesis system used. For example, in the case of a systemusing a wheat-derived germ extract, the reaction may be carried outusually at 10° C. to 40° C., preferably 18° C. to 30° C., morepreferably 20° C. to 26° C., for 10 minutes to 48 hours, preferably for10 minutes to 30 hours, more preferably for 10 minutes to 24 hours.

Since the mRNAs coding for a CPT family protein, a NgBR family protein,and a REF family protein contained in the cell-free protein synthesissolution are easily broken down, the mRNAs may be additionally added asappropriate during the protein synthesis reaction to make the proteinsynthesis more efficient. Thus, in another suitable embodiment of thefirst invention, the mRNAs coding for a CPT family protein, a NgBRfamily protein, and a REF family protein are additionally added duringthe protein synthesis reaction.

The addition time, the number of additions, the addition amount andother conditions of the mRNAs are not particularly limited, and may beselected appropriately.

In the production method of the first invention, a step of collectingthe rubber particles may optionally be performed after the step ofbinding a protein expressed by a gene coding for a cis-prenyltransferase(CPT) family protein, a protein expressed by a gene coding for a Nogo-Breceptor (NgBR) family protein, and a protein expressed by a gene codingfor a rubber elongation factor (REF) family protein to rubber particlesin vitro.

The rubber particle collection step may be carried out by any method,provided that the rubber particles can be collected. It may be carriedout by conventional methods for collecting rubber particles. Specificexamples include methods using centrifugation. When the rubber particlesare collected by the centrifugation, the centrifugal force,centrifugation time, and centrifugation temperature may be selectedappropriately so as to allow the rubber particles to be collected. Forexample, the centrifugal force during the centrifugation is preferably15000×g or more, more preferably 20000×g or more, still more preferably25000×g or more. Since increasing the centrifugal force too much is notexpected to produce a correspondingly high separation effect, the upperlimit of the centrifugal force is preferably 50000×g or less, morepreferably 45000×g or less. The centrifugation time is preferably atleast 20 minutes, more preferably at least 30 minutes, still morepreferably at least 40 minutes. Since increasing the centrifugation timetoo much is not expected to produce a correspondingly high separationeffect, the upper limit of the centrifugation time is preferably 120minutes or less, more preferably 90 minutes or less.

From the standpoint of maintaining the protein activity of the CPTfamily protein, NgBR family protein, and REF family protein bound to therubber particles, the centrifugation temperature is preferably 0° C. to10° C., more preferably 2° C. to 8° C., particularly preferably 4° C.

For example, when the cell-free protein synthesis is performed, therubber particles and the cell-free protein synthesis solution areseparated into the upper layer and the lower layer, respectively, by thecentrifugation. The cell-free protein synthesis solution as the lowerlayer may then be removed to collect the rubber particles bound to theCPT family protein, NgBR family protein, and REF family protein. Thecollected rubber particles may be re-suspended in a suitable buffer witha neutral pH for storage.

Since the CPT family protein, NgBR family protein, and REF familyprotein to be bound to the rubber particles are proteins that inherentlyexist on rubber particles in rubber-producing plants, the rubberparticles collected by the rubber particle collection step can be usedin the same way as usual natural rubber without the need for furtherspecial treatment.

Moreover, the polyisoprenoid obtained by the method for producing apolyisoprenoid of the first invention can be collected by subjecting therubber particles to the following solidification step.

The method for solidification is not particularly limited, and examplesinclude a method of adding the rubber particles to a solvent that doesnot dissolve the polyisoprenoid (natural rubber), such as ethanol,methanol or acetone; and a method of adding an acid to the rubberparticles. Rubber (natural rubber) can be recovered as solids from therubber particles by the solidification step. The obtained rubber(natural rubber) may be dried as necessary before use.

As described above, according to the first invention, the rubbersynthesis activity of rubber particles can be enhanced by binding aprotein expressed by a gene coding for a cis-prenyltransferase (CPT)family protein, a protein expressed by a gene coding for a Nogo-Breceptor (NgBR) family protein, and a protein expressed by a gene codingfor a rubber elongation factor (REF) family protein to rubber particlesin vitro. Thus, it is possible to produce rubber (one example ofpolyisoprenoids) more efficiently in reaction vessels (e.g. test tubes,industrial plants).

Thus, another aspect of the first invention relates to a method forsynthesizing a polyisoprenoid, which includes a step of binding aprotein expressed by a gene coding for a cis-prenyltransferase (CPT)family protein, a protein expressed by a gene coding for a Nogo-Breceptor (NgBR) family protein, and a protein expressed by a gene codingfor a rubber elongation factor (REF) family protein to rubber particlesin vitro, for example in a reaction vessel (e.g. a test tube orindustrial plant).

The step of binding a protein expressed by a gene coding for acis-prenyltransferase (CPT) family protein, a protein expressed by agene coding for a Nogo-B receptor (NgBR) family protein, and a proteinexpressed by a gene coding for a rubber elongation factor (REF) familyprotein to rubber particles in vitro is as described above.

Herein, the term “polyisoprenoid” is a collective term for polymerscomposed of isoprene units (C₅H₈). Examples of the polyisoprenoidinclude sesterterpenes (C₂₅), triterpenes (C₃₀), tetraterpenes (C₄₀),natural rubber, and other polymers. Herein, the term “isoprenoid” refersto a compound having isoprene units (C₅H₈), and conceptually includespolyisoprenoids.

(Method for Producing Rubber Product)

The method for producing a rubber product of the first inventionincludes the steps of: kneading a polyisoprenoid produced by the methodfor producing a polyisoprenoid of the first invention with an additiveto obtain a kneaded mixture; forming a raw rubber product from thekneaded mixture; and vulcanizing the raw rubber product.

The rubber product is not particularly limited as long as it is a rubberproduct that can be produced from rubber, preferably natural rubber, andexamples include pneumatic tires, rubber rollers, rubber fenders,gloves, and medical rubber tubes.

When the rubber product is a pneumatic tire, or in other words when themethod for producing a rubber product of the first invention is a methodfor producing a pneumatic tire, the raw rubber product forming stepcorresponds to a green tire building step in which a green tire is builtfrom the kneaded mixture, and the vulcanization step corresponds to avulcanization step in which the green tire is vulcanized. Thus, themethod for producing a pneumatic tire of the first invention includesthe steps of: kneading a polyisoprenoid produced by the method forproducing a polyisoprenoid with an additive to obtain a kneaded mixture;building a green (or raw) tire from the kneaded mixture; and vulcanizingthe green tire.

<Kneading Step>

In the kneading step, the polyisoprenoid produced by the method forproducing a polyisoprenoid is kneaded with an additive to obtain akneaded mixture.

The additive is not particularly limited, and additives used inproduction of rubber products may be used. For example, when the rubberproduct is a pneumatic tire, examples include rubber components otherthan the polyisoprenoid, reinforcing fillers such as carbon black,silica, calcium carbonate, alumina, clay, or talc, silane couplingagents, zinc oxide, stearic acid, processing aids, various antioxidants,softeners such as oil, wax, vulcanizing agents such as sulfur, andvulcanization accelerators.

The kneading in the kneading step may be carried out using an open rollmill, a Banbury mixer, an internal mixer, or other rubber kneadingmachines.

<Raw Rubber Product Forming Step (Green Tire Building Step for Tire)>

In the raw rubber product forming step, a raw rubber product (green tirefor tire) is formed (or built) from the kneaded mixture obtained in thekneading step.

The method for forming a raw rubber product is not particularly limited,and methods used to form raw rubber products may be used appropriately.For example, when the rubber product is a pneumatic tire, the kneadedmixture obtained in the kneading step may be extruded according to theshape of a tire component and then formed by a usual method on a tirebuilding machine and assembled with other tire components to build agreen tire (unvulcanized tire).

<Vulcanization Step>

In the vulcanization step, the raw rubber product obtained in the rawrubber product forming step is vulcanized to obtain a rubber product.

The method for vulcanizing the raw rubber product is not particularlylimited, and methods used to vulcanize raw rubber products may be usedappropriately. For example, when the rubber product is a pneumatic tire,the green tire (unvulcanized tire) obtained in the green tire buildingstep may be vulcanized by heating and pressing in a vulcanizer to obtaina pneumatic tire.

(Second Invention)

The method for producing a polyisoprenoid of the second inventionincludes producing a polyisoprenoid in a transformed plant produced byintroducing a gene coding for a cis-prenyltransferase (CPT) familyprotein, a gene coding for a Nogo-B receptor (NgBR) family protein, anda gene coding for a rubber elongation factor (REF) family protein into aplant to allow the plant to express the CPT family protein, the NgBRfamily protein, and the REF family protein.

The inventors were the first to discover that the rubber synthesis ofrubber particles is activated by binding a CPT family protein, a NgBRfamily protein, and a REF family protein to rubber particles in vitro.Based on this finding, it is expected that rubber synthesis activity canbe enhanced by co-expressing a CPT family protein, a NgBR familyprotein, and a REF family protein in a plant. Thus, the use of atransformed plant engineered to co-express a CPT family protein, a NgBRfamily protein, and a REF family protein in polyisoprenoid production isexpected to result in increased polyisoprenoid production.

It is also considered that rubber synthesis activity can be improved intransformed plants produced by introducing a gene coding for a CPTfamily protein, a gene coding for a NgBR family protein, and a genecoding for a REF family protein individually into respective plants.However, the co-expression of these three genes is considered to providea further synergistic effect. For example, when the CPT family proteinis expressed alone, if the amount of the NgBR family protein expressedis low, the CPT family protein may fail to bind to rubber particles and,therefore, fail to function. Also, when the NgBR family protein isexpressed alone, if the amount of the CPT family protein (synthase)expressed is low, the rubber synthesis activity may not be sufficientlyimproved. In contrast, co-expression of the CPT family protein and NgBRfamily protein is considered to lead to enhanced rubber synthesisactivity, and when the REF family protein is further expressed, it isconsidered that the rubber particles that are expected to have enhancedrubber synthesis activity due to the co-expression of the CPT familyprotein and NgBR family protein can stably accumulate in the plant.Thus, it is expected that rubber synthesis activity can be significantlyenhanced by co-expressing the CPT family protein, NgBR family protein,and REF family protein in a plant.

Since the method for producing a polyisoprenoid of the second inventionuses a transformed plant having specific genes introduced therein,unlike conventional methods involving chemical administration,continuous effects can be expected without subsequent continuingtreatment because once the genes have been introduced, the effects ofthe gene introduction are obtained through inherent biologicalmechanisms.

The origins of the gene coding for a cis-prenyltransferase (CPT) familyprotein, the gene coding for a Nogo-B receptor (NgBR) family protein,and the gene coding for a rubber elongation factor (REF) family proteinare not particularly limited, but preferably they are each derived fromany of the plants described above, more preferably at least one selectedfrom the group consisting of plants of the genera Hevea, Sonchus,Taraxacum, and Parthenium. Among these, they are each still morepreferably derived from at least one species of plant selected from thegroup consisting of Hevea brasiliensis, Sonchus oleraceus, Partheniumargentatum, and Taraxacum kok-saghyz, particularly preferably Heveabrasiliensis. Most preferably, they are all derived from Heveabrasiliensis. Whatever their origins, the gene coding for a CPT familyprotein, the gene coding for a NgBR family protein, and the gene codingfor a REF family protein are preferably derived from the species ofplant into which they are to be introduced.

The gene coding for a cis-prenyltransferase (CPT) family protein, thegene coding for a Nogo-B receptor (NgBR) family protein, and the genecoding for a rubber elongation factor (REF) family protein, and the CPTfamily protein, the NgBR family protein, and the REF family protein usedin the second invention are as described above in connection with thefirst invention.

In the method for producing a polyisoprenoid of the second invention, agene coding for another protein may further be introduced into the plantas long as the gene coding for a cis-prenyltransferase (CPT) familyprotein, the gene coding for a Nogo-B receptor (NgBR) family protein,and the gene coding for a rubber elongation factor (REF) family proteinare introduced into the plant.

The gene coding for another protein may be as described above inconnection with the first invention.

By introducing the gene coding for a cis-prenyltransferase (CPT) familyprotein, the gene coding for a Nogo-B receptor (NgBR) family protein,and the gene coding for a rubber elongation factor (REF) family proteininto a plant, a plant (transformed plant) is produced which has beentransformed to express the CPT family protein, the NgBR family protein,and the REF family protein. Since the CPT family protein, NgBR familyprotein, and REF family protein are co-expressed in the transformedplant, the activity of the CPT family protein is expected to bestabilized and enhanced. Therefore, it is expected that the transformedplant engineered to co-express the CPT family protein, NgBR familyprotein, and REF family protein exhibits continuously enhanced rubbersynthesis activity, and the use of such a transformed plant inpolyisoprenoid production can suitably result in an increase inpolyisoprenoid production.

The method for preparing the plant transformed to express the CPT familyprotein, NgBR family protein, and REF family protein (transformed plant)is explained briefly below, but such a transformed plant can be preparedby conventionally known methods.

Specifically, for example, the method for preparing the transformedplant may be carried out as follows: A DNA containing the nucleotidesequence of SEQ ID NO:1, a DNA containing the nucleotide sequence of SEQID NO:3, and a DNA containing the nucleotide sequence of SEQ ID NO:5 areinserted downstream of the promoter of a suitable expression vector withsuitable restriction enzymes and the like to prepare a recombinant DNA.This recombinant DNA may then be introduced into host plant cells whichare compatible with the expression vector, to obtain transformed plantcells. Alternatively, an expression vector in which a DNA containing thenucleotide sequence of SEQ ID NO:1 is inserted downstream of thepromoter with suitable restriction enzymes and the like, an expressionvector in which a DNA containing the nucleotide sequence of SEQ ID NO:3is inserted downstream of the promoter with suitable restriction enzymesand the like, and an expression vector in which a DNA containing thenucleotide sequence of SEQ ID NO:5 is inserted downstream of thepromoter with suitable restriction enzymes and the like are used toprepare recombinant DNAs, and these recombinant DNAs may then beintroduced into host plant cells which are compatible with theexpression vectors, to obtain transformed plant cells.

There are no particular restrictions on the plant (host plant cells)into which the recombinant DNA is to be introduced, but since improvedpolyisoprenoid productivity and increased polyisoprenoid production canbe expected in particular when the CPT family protein, NgBR familyprotein, and REF family protein are expressed in plants capable ofpolyisoprenoid biosynthesis, the plant is preferably a rubber-producingplant, and the host plant cells are preferably plant cells of arubber-producing plant. Thus, in another suitable embodiment of thesecond invention, the plant into which the gene coding for acis-prenyltransferase (CPT) family protein, the gene coding for a Nogo-Breceptor (NgBR) family protein, and the gene coding for a rubberelongation factor (REF) family protein are to be introduced is apolyisoprenoid-producing plant, more preferably at least one species ofrubber-producing plant selected from the group consisting of Heveabrasiliensis, Sonchus oleraceus, Parthenium argentatum, and Taraxacumkok-saghyz, particularly preferably Hevea brasiliensis.

The expression vector may be a vector that is capable of autonomousreplication in the host plant cells or can be incorporated into thechromosome, and, further, contains a promoter at a position allowingtranscription of the recombinant DNA.

Examples of the expression vector include pBI vectors, Ti plasmids, andtobacco mosaic virus vectors.

Any promoter that functions in plant cells can be used as the promoter,and examples include cauliflower mosaic virus (CaMV) 35S promoter, riceactin 1 promoter, nopaline synthase gene promoter, tobacco mosaic virus35S promoter, and rice-derived actin gene promoter.

It is preferred to use an expression vector carrying a promoter that isspecific to a tissue in which an isoprenoid compound is biosynthesized,such as lactiferous ducts. Plant growth retardation and other harmfuleffects can be reduced by expressing specifically in a tissue in whichan isoprenoid is biosynthesized.

Any method for introducing DNA into host plant cells may be used tointroduce the recombinant DNA, and examples include methods usingAgrobacterium (JP S59-140885 A, JP S60-70080 A, WO94/00977, which areincorporated herein by reference), electroporation methods (JPS60-251887 A, incorporated herein by reference), and methods usingparticle guns (JP 2606856 B, JP 2517813 B, which are incorporated hereinby reference).

The transformed plant (transformed plant cells) can be obtained by theabove or other methods. The transformed plant conceptually includes notonly transformed plant cells obtained by the above methods but also alltheir progeny or clones and even progeny plants obtained by passagingthese cells. Once transformed plant cells into which the DNAs or thevector(s) have been introduced in the genome are obtained, progeny orclones can be obtained from the transformed plant cells by sexual orasexual reproduction, tissue culture, cell culture, cell fusion, orother techniques. Further, the transformed plant cells, or progeny orclones thereof may be used to obtain reproductive materials (e.g. seeds,fruits, cuttings, stem tubers, root tubers, shoots, adventitious buds,adventitious embryos, calluses, protoplasts), which can then be used toproduce the transformed plant on a large scale.

Techniques to regenerate plants (transformed plants) from thetransformed plant cells are already known; for example, Doi et al.disclose techniques for eucalyptus (JP H11-127025), Fujimura et al.disclose techniques for rice (Fujimura et al., (1995), Plant TissueCulture Lett., vol. 2: p 74-), Shillito et al. disclose techniques forcorn (Shillito et al., (1989), Bio/Technology, vol. 7: p 581-), Visseret al. disclose techniques for potato (Visser et al., (1989), Theor.Appl. Genet., vol. 78: p 589-), and Akama et al. disclose techniques forArabidopsis thaliana (Akama et al., (1992), Plant Cell Rep., vol. 12: p7-)(all the above documents are incorporated herein by reference). Thoseskilled in the art can regenerate plants from the transformed plantcells according to these documents.

The expression of the target protein genes in the regenerated plant canbe confirmed by known techniques. For example, the expression of thetarget proteins may be analyzed by Western blot analysis.

Seeds may be obtained from the transformed plant as follows: Forexample, the transformed plant is rooted in a suitable medium, and therooted plant is then transplanted into a pot filled with soil containingwater. This plant is grown under suitable cultivation conditions untilit finally forms seeds, to obtain the seeds. Moreover, plants may beobtained from the seeds as follows: For example, the seeds from thetransformed plant obtained as described above may be sown in soilcontaining water and grown under suitable cultivation conditions toobtain plants.

In the second invention, it is expected that polyisoprenoid productivitycan be improved by performing polyisoprenoid production using thetransformed plant having introduced therein a gene coding for a CPTfamily protein, a gene coding for a NgBR family protein, and a genecoding for a REF family protein. Specifically, polyisoprenoid productionmay be carried out by culturing the transformed plant cells obtained asdescribed above, calluses obtained from the transformed plant cells,cells re-differentiated from the calluses, or the like in a suitablemedium, or by growing transformed plants re-differentiated from thetransformed plant cells, plants obtained from seeds obtained from thetransformed plants, or the like under suitable cultivation conditions.

The polyisoprenoid produced in the method for producing a polyisoprenoidof the second invention can be obtained by harvesting latex from thetransformed plant, and subjecting the harvested latex to the followingsolidification step.

The method for harvesting latex from the transformed plant is notparticularly limited, and ordinary harvesting methods may be used. Forexample, latex may be harvested by collecting the emulsion oozing outfrom the cuts in the trunk of the plant (tapping), or the emulsionoozing out from the cut roots or other parts of the transformed plant,or by crushing the cut tissue followed by extraction with an organicsolvent.

In the solidification step, the method for solidification is notparticularly limited, and examples include a method of adding the latexto a solvent that does not dissolve the polyisoprenoid (natural rubber),such as ethanol, methanol or acetone; and a method of adding an acid tothe latex. Rubber (natural rubber) can be recovered as solids from thelatex by the solidification step. The obtained rubber (natural rubber)may be dried as necessary before use.

Thus, another aspect of the second invention relates to a transformedplant produced by the method for producing a polyisoprenoid of thesecond invention. The CPT family protein activity in the transformedplant of the second invention is expected to be stabilized and enhancedby the introduced proteins. Therefore, it is expected that thetransformed plant exhibits continuously enhanced rubber synthesisactivity, and the use of such a transformed plant in polyisoprenoidproduction can result in an increase in polyisoprenoid production.

The gene coding for a cis-prenyltransferase (CPT) family protein, thegene coding for a Nogo-B receptor (NgBR) family protein, and the genecoding for a rubber elongation factor (REF) family protein in thetransformed plant of the second invention, and the plant into whichthese genes are to be introduced are all as described above.

(Method for Producing Rubber Product)

The method for producing a rubber product of the second inventionincludes the steps of: kneading a polyisoprenoid produced by the methodfor producing a polyisoprenoid of the second invention with an additiveto obtain a kneaded mixture; forming a raw rubber product from thekneaded mixture; and vulcanizing the raw rubber product.

The rubber product is as described above in connection with the firstinvention.

When the rubber product is a pneumatic tire, or in other words when themethod for producing a rubber product of the second invention is amethod for producing a pneumatic tire, the raw rubber product formingstep corresponds to a green tire building step in which a green tire isbuilt from the kneaded mixture, and the vulcanization step correspondsto a vulcanization step in which the green tire is vulcanized. Thus, themethod for producing a pneumatic tire of the second invention includesthe steps of: kneading a polyisoprenoid produced by the method forproducing a polyisoprenoid of the second invention with an additive toobtain a kneaded mixture; building a green (or raw) tire from thekneaded mixture; and vulcanizing the green tire.

<Kneading Step>

The kneading step is as described above in connection with the firstinvention.

<Raw Rubber Product Forming Step (Green Tire Building Step for Tire)>

The raw rubber product forming step is as described above in connectionwith the first invention.

<Vulcanization Step>

The vulcanization step is as described above in connection with thefirst invention.

EXAMPLES

The present invention is specifically explained with reference toexamples, but the present invention is not limited to these examples.

Example 1 Extraction of Total RNA from Hevea Latex

Total RNA was extracted from the latex of Hevea brasiliensis by the hotphenol method. To 6 mL of the latex were added 6 mL of 100 mM sodiumacetate buffer and 1 mL of a 10% SDS solution, and then 12 mL ofwater-saturated phenol pre-heated at 65° C. The mixture was incubatedfor 5 minutes at 65° C., agitated in a vortex, and centrifuged at 7000rpm for 10 minutes at room temperature. After the centrifugation, thesupernatant was transferred to a new tube, 12 mL of a phenol:chloroform(1:1) solution was added, and the mixture was agitated by shaking for 2minutes. After the agitation, the resulting mixture was centrifugedagain at 7000 rpm for 10 minutes at room temperature, the supernatantwas transferred to a new tube, 12 mL of a chloroform:isoamyl alcohol(24:1) solution was added, and the mixture was agitated by shaking for 2minutes. After the agitation, the resulting mixture was centrifugedagain at 7000 rpm for 10 minutes at room temperature, the supernatantwas transferred to a new tube, 1.2 mL of a 3M sodium acetate solutionand 13 mL of isopropanol were added, and the mixture was agitated in avortex. The resulting mixture was incubated for 30 minutes at −20° C. toprecipitate total RNA. The incubated mixture was centrifuged at 15000rpm for 10 minutes at 4° C., and the supernatant was removed to collecta precipitate of total RNA. The collected total RNA was washed twicewith 70% ethanol, and dissolved in RNase-free water.

[Synthesis of cDNA from Total RNA]

cDNA was synthesized from the collected total RNA. The cDNA synthesiswas carried out using a PrimeScript II 1st strand cDNA synthesis kit(Takara) in accordance with the manual.

[Acquisition of CPT, NgBR and REF Genes from cDNA]

CPT, NgBR and REF genes were obtained using the prepared 1st strand cDNAas a template. PCR was performed using a KOD-plus-Neo (Toyobo Co., Ltd.)in accordance with the manual. The PCR reaction involved 35 cycles witheach cycle consisting of 10 seconds at 98° C., 30 seconds at 58° C., and1 minute at 68° C.

The CPT gene was obtained using the following primers:

Primer 1: 5′-tttggatccgatggaattatacaacggtgagagg-3′, Primer 2:5′-tttgcggccgcttattttaagtattccttatgtttctcc-3′.

The NgBR gene was obtained using the following primers:

Primer 3: 5′-tttctcgagatggatttgaaacctggagctg-3′, Primer 4:5′-tttctcgagtcatgtaccataattttgctgcac-3′.

The REF gene was obtained using the following primers:

Primer 5: 5′-tttctcgagatggctgaagacgaagac-3′, Primer 6:5′-tttggatcctcaattctctccataaaac-3′.

CPT gene (HRT1), NgBR gene (HRTBP), and REF gene were obtained as above.The genes were sequenced to identify the full-length nucleotide sequenceand amino acid sequence. The nucleotide sequence of HRT1 is given by SEQID NO:1. The amino acid sequence of HRT1 is given by SEQ ID NO:2. Thenucleotide sequence of HRTBP is given by SEQ ID NO:3. The amino acidsequence of HRTBP is given by SEQ ID NO:4. The nucleotide sequence ofREF is given by SEQ ID NO:5. The amino acid sequence of REF is given bySEQ ID NO:6.

[Vector Construction]

The obtained DNA fragments were subjected to dA addition and theninserted into pGEM-T Easy vectors using a pGEM-T Easy Vector System(Promega) to prepare pGEM-HRT1, pGEM-HRTBP, and pGEM-REF.

[Transformation of E. coli]

E. coli DH5α was transformed with the prepared vectors, the transformantwas cultured on LB agar medium containing ampicillin and X-gal, and E.coli cells carrying the introduced target genes were selected byblue/white screening.

[Plasmid Extraction]

The E. coli cells transformed with the plasmids containing the targetgenes were cultured overnight at 37° C. on LB liquid medium. After theculture, the cells were collected, and the plasmids were collected. AFastGene Plasmid mini kit (Nippon Genetics Co., Ltd.) was used forplasmid collection.

It was confirmed by sequence analysis that there were no mutations inthe nucleotide sequences of the genes inserted into the collectedplasmids.

[Preparation of Vectors for Cell-Free Protein Synthesis]

The pGEM-HRT1 obtained in the above “Vector construction” was treatedwith the restriction enzymes Bam HI and Not I, and inserted into apEU-E01-His-TEV-MCS-N2 cell-free expression vector that had been treatedsimilarly with Bam HI and Not I, to prepare pEU-His-N2-HRT1.

Similarly, pGEM-HRTBP was treated with the restriction enzyme Xho I, andinserted into a pEU-E01-MCS-TEV-His-C1 cell-free expression vector thathad been treated similarly with Xho I, to prepare pEU-C1-HRTBP.

Furthermore, pGEM-REF was treated with the restriction enzymes Xho I andBam HI, and inserted into a pEU-E01-MCS-TEV-His-C1 cell-free expressionvector that had been treated similarly with Xho I and Bam HI, to preparepEU-C1-REF.

[Transformation of E. coli]

E. coli DH5α was transformed with the prepared vectors, the transformantwas cultured on LB agar medium containing ampicillin and X-gal, and E.coli cells carrying the introduced target genes were selected by colonyPCR.

[Plasmid Extraction]

The E. coli cells transformed with the plasmids containing the targetgenes were cultured overnight at 37° C. on LB liquid medium. After theculture, the cells were collected, and the plasmids were collected. AFastGene Plasmid mini kit (Nippon Genetics Co., Ltd.) was used forplasmid collection.

[Preparation of Rubber Particles]

Rubber particles were prepared from Hevea latex by five stages ofcentrifugation. To 900 mL of Hevea latex was added 100 mL of 1 M Trisbuffer (pH 7.5) containing 20 mM dithiothreitol (DTT) to prepare a latexsolution. The latex solution was centrifuged in stages at the followingdifferent speeds: 1000×g, 2000×g, 8000×g, 20000×g, and 50000×g. Eachstage of centrifugation was carried out for 45 minutes at 4° C. To therubber particle layer remaining after the centrifugation at 50000×g wasadded 3-[(3-cholamidopropyl)dimethylamino]-propanesulfonate (CHAPS) at afinal concentration of 0.1 to 2.0×CMC (0.1 to 2.0 times the criticalmicelle concentration CMC) to wash the rubber particles. After thewashing, the rubber particles were collected by ultracentrifugation(40000×g, 4° C., 45 minutes), and re-suspended in an equal amount of 100M Tris buffer (pH 7.5) containing 2 mM dithiothreitol (DTT).

[Cell-Free Protein Synthesis Reaction (Step 1: mRNA TranscriptionReaction)]

Cell-free protein synthesis was performed using a WEPRO7240H expressionkit (CellFree Sciences Co., Ltd.). mRNA transcription reactions wereperformed using the vectors obtained in the above “Preparation ofvectors for cell-free protein synthesis” as templates in accordance withthe protocol of the WEPRO7240H expression kit.

[Purification of mRNAs]

After the transcription reactions, the resulting mRNAs were purified byethanol precipitation.

[Cell-Free Protein Synthesis Reaction (Step 2: Protein Synthesis byDialysis)]

The following amounts were added to a dialysis cup (MWCO 12000,Bio-Teck). A total amount of 60 μL of a reaction solution was preparedaccording to the protocol of the WEPRO7240H expression kit. To thereaction solution was added 1 to 2 mg of the rubber particles.Separately, 650 μL of SUB-AMIX was added to a No. 2 PP container(Maruemu container).

The dialysis cup was set in the No. 2 PP container, and a proteinsynthesis reaction was initiated at 26° C. The addition of mRNAs and thereplacement of the outer dialysis solution (SUB-AMIX) were performedtwice after the initiation of the reaction.

The reaction was carried out for 24 hours. A schematic diagramillustrating the dialysis process is shown in FIG. 3.

[Collection of Rubber Particles after Reaction]

The solution in the dialysis cup was transferred to a new 1.5 μL tube,and the reacted rubber particles were collected by ultracentrifugation(40000×g, 4° C., 45 minutes) and re-suspended in an equal amount of 100M Tris buffer (pH 7.5) containing 2 mM dithiothreitol (DTT).

[Measurement of Rubber Synthesis Activity of Reacted Rubber Particles]

The rubber synthesis activity of the rubber particles collected afterthe reaction was measured as follows.

First, 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 5 mM MgCl₂, 15 μM farnesyldiphosphate (FPP), 100 μM 1-14C isopentenyl diphosphate ([1-14C]IPP,specific activity 5 Ci/mol), and 10 μL of the rubber particle solutionwere mixed to prepare a reaction solution (100 L in total), which wasthen reacted for 16 hours at 30° C.

After the reaction, 200 L of saturated NaCl was added to the solution,and the mixture was extracted with 1 mL of diethyl ether to extractisopentenol and the like. Next, polyprenyl diphosphates were extractedfrom the aqueous phase with 1 mL of BuOH saturated with saline, and thenan ultra-long-chain polyisoprenoid (natural rubber) was furtherextracted from the aqueous phase with 1 mL of toluene/hexane (1:1),followed by determination of radioactivity. The radioactivity wasdetermined by ¹⁴C counting using a liquid scintillation counter. Ahigher radioactivity (dpm) indicates higher natural rubber productionand higher rubber synthesis activity.

The results are shown in Table 1.

Comparative Example 1 Preparation of Rubber Particles

Rubber particles were prepared as in Example 1.

[Cell-Free Protein Synthesis Reaction (Step 1: mRNA TranscriptionReaction)]

Cell-free protein synthesis was performed using a WEPRO7240H expressionkit (CellFree Sciences Co., Ltd.). An mRNA transcription reaction wasperformed using the cell-free expression vector pEU-E01-His-TEV-MCS-N2as a template in accordance with the protocol of the WEPRO7240Hexpression kit.

[Purification of mRNA]

After the transcription reaction, the resulting mRNA was purified byethanol precipitation.

[Cell-Free Protein Synthesis Reaction (Step 2: Protein Synthesis byDialysis)]

The same procedure as in Example 1 was followed but using the abovemRNA.

[Collection of Rubber Particles after Reaction]

The reacted rubber particles were collected as in Example 1, andre-suspended in an equal amount of 100 M Tris buffer (pH 7.5) containing2 mM dithiothreitol (DTT).

[Measurement of Rubber Synthesis Activity of Reacted Rubber Particles]

The rubber synthesis activity of the rubber particles collected afterthe reaction was measured as in Example 1.

Comparative Example 2

The same procedure as in Example 1 was followed but using the pEU-C1-REFobtained in the above “Preparation of vectors for cell-free proteinsynthesis” in Example 1 as the template for cell-free protein synthesis,and the rubber synthesis activity of the rubber particles collectedafter the reaction was measured as in Example 1.

The results are shown in Table 1.

Comparative Example 3

The same procedure as in Example 1 was followed but using thepEU-C1-HRTBP obtained in the above “Preparation of vectors for cell-freeprotein synthesis” in Example 1 as the template for cell-free proteinsynthesis, and the rubber synthesis activity of the rubber particlescollected after the reaction was measured as in Example 1.

The results are shown in Table 1.

Comparative Example 4

The same procedure as in Example 1 was followed but using thepEU-His-N2-HRT1 obtained in the above “Preparation of vectors forcell-free protein synthesis” in Example 1 as the template for cell-freeprotein synthesis, and the rubber synthesis activity of the rubberparticles collected after the reaction was measured as in Example 1.

The results are shown in Table 1.

TABLE 1 Radioac- Bound tivity protein (dpm) Comparative None  7500Example 1 Comparative REF  6000 Example 2 Comparative HRTBP  5800Example 3 Comparative HRT1 15000 Example 4 Example 1 HRT1 + HRTBP +25000 REF

Table 1 shows that by binding a CPT family protein, a NgBR familyprotein, and a REF family protein to rubber particles, the rubbersynthesis activity of rubber particles was significantly enhanced ascompared to when these proteins were bound alone to rubber particles.Further, in Comparative Example 2 in which REF was bound alone to rubberparticles and Comparative Example 3 in which NgBR was bound alone torubber particles, the rubber synthesis activity was lower than inComparative Example 1 with no bound proteins. From these results itseems that the combination of a CPT family protein, a NgBR familyprotein, and a REF family protein has a synergistic effect that isgreater than the sum of their individual effects. Thus, the rubbersynthesis activity of rubber particles is significantly enhanced by thespecific combination of a CPT family protein, a NgBR family protein, anda REF family protein, and this effect could not be predicted even bythose skilled in the art.

(Sequence Listing Free Text)

SEQ ID NO:1: Nucleotide sequence of gene coding for HRT1 from Heveabrasiliensis

SEQ ID NO:2: Amino acid sequence of HRT1 from Hevea brasiliensis

SEQ ID NO:3: Nucleotide sequence of gene coding for HRTBP from Heveabrasiliensis

SEQ ID NO:4: Amino acid sequence of HRTBP from Hevea brasiliensis

SEQ ID NO:5: Nucleotide sequence of gene coding for REF from Heveabrasiliensis

SEQ ID NO:6: Amino acid sequence of REF from Hevea brasiliensis

SEQ ID NO:7: Primer 1

SEQ ID NO:8: Primer 2

SEQ ID NO:9: Primer 3

SEQ ID NO:10: Primer 4

SEQ ID NO:11: Primer 5

SEQ ID NO:12: Primer 6

1. A method for producing a polyisoprenoid, the method comprising: astep of binding a protein expressed by a gene coding for acis-prenyltransferase (CPT) family protein, a protein expressed by agene coding for a Nogo-B receptor (NgBR) family protein, and a proteinexpressed by a gene coding for a rubber elongation factor (REF) familyprotein to rubber particles in vitro.
 2. The method for producing apolyisoprenoid according to claim 1, wherein at least one selected fromthe group consisting of the gene coding for a cis-prenyltransferase(CPT) family protein, the gene coding for a Nogo-B receptor (NgBR)family protein, and the gene coding for a rubber elongation factor (REF)family protein is derived from a plant.
 3. The method for producing apolyisoprenoid according to claim 2, wherein at least one selected fromthe group consisting of the gene coding for a cis-prenyltransferase(CPT) family protein, the gene coding for a Nogo-B receptor (NgBR)family protein, and the gene coding for a rubber elongation factor (REF)family protein is derived from Hevea brasiliensis.
 4. The method forproducing a polyisoprenoid according to claim 1, wherein the bindingstep comprises performing protein synthesis in the presence of bothrubber particles and a cell-free protein synthesis solution containingan mRNA coding for a cis-prenyltransferase (CPT) family protein, an mRNAcoding for a Nogo-B receptor (NgBR) family protein, and an mRNA codingfor a rubber elongation factor (REF) family protein to bind the CPTfamily protein, the NgBR family protein, and the REF family protein tothe rubber particles.
 5. The method for producing a polyisoprenoidaccording to claim 4, wherein the cell-free protein synthesis solutioncontains a germ extract.
 6. The method for producing a polyisoprenoidaccording to claim 5, wherein the germ extract is derived from wheat. 7.The method for producing a polyisoprenoid according to claim 4, whereinthe rubber particles are present in the cell-free protein synthesissolution at a concentration of 5 to 50 g/L.
 8. A method for producing apolyisoprenoid, the method comprising producing a polyisoprenoid in atransformed plant produced by introducing a gene coding for acis-prenyltransferase (CPT) family protein, a gene coding for a Nogo-Breceptor (NgBR) family protein, and a gene coding for a rubberelongation factor (REF) family protein into a plant to allow the plantto express the CPT family protein, the NgBR family protein, and the REFfamily protein.
 9. The method for producing a polyisoprenoid accordingto claim 8, wherein at least one selected from the group consisting ofthe gene coding for a cis-prenyltransferase (CPT) family protein, thegene coding for a Nogo-B receptor (NgBR) family protein, and the genecoding for a rubber elongation factor (REF) family protein is derivedfrom a plant.
 10. The method for producing a polyisoprenoid according toclaim 9, wherein at least one selected from the group consisting of thegene coding for a cis-prenyltransferase (CPT) family protein, the genecoding for a Nogo-B receptor (NgBR) family protein, and the gene codingfor a rubber elongation factor (REF) family protein is derived fromHevea brasiliensis.
 11. The method for producing a polyisoprenoidaccording to claim 8, wherein the gene coding for acis-prenyltransferase (CPT) family protein is the following DNA [1] or[2]: [1] a DNA comprising the nucleotide sequence of SEQ ID NO:1; or [2]a DNA that hybridizes under stringent conditions with a DNA comprising anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO:1, and codes for a protein having an enzyme activity that catalyzes areaction of cis-chain elongation of an isoprenoid compound.
 12. Themethod for producing a polyisoprenoid according to claim 8, wherein thegene coding for a Nogo-B receptor (NgBR) family protein is the followingDNA [3] or [4]: [3] a DNA comprising the nucleotide sequence of SEQ IDNO:3; or [4] a DNA that hybridizes under stringent conditions with a DNAcomprising a nucleotide sequence complementary to the nucleotidesequence of SEQ ID NO:3, and codes for a protein having the functions ofbinding to a membrane via one or more transmembrane domains on theN-terminal side of the protein, and interacting with another protein onthe C-terminal side thereof.
 13. The method for producing apolyisoprenoid according to claim 8, wherein the gene coding for arubber elongation factor (REF) family protein is the following DNA [5]or [6]: [5] a DNA comprising the nucleotide sequence of SEQ ID NO:5; or[6] a DNA that hybridizes under stringent conditions with a DNAcomprising a nucleotide sequence complementary to the nucleotidesequence of SEQ ID NO:5, and codes for a rubber particle-associatedprotein that is bound to rubber particles in latex.
 14. A transformedplant, produced by introducing a gene coding for a cis-prenyltransferase(CPT) family protein, a gene coding for a Nogo-B receptor (NgBR) familyprotein, and a gene coding for a rubber elongation factor (REF) familyprotein into a plant to allow the plant to express the CPT familyprotein, the NgBR family protein, and the REF family protein.
 15. Thetransformed plant according to claim 14, wherein at least one selectedfrom the group consisting of the gene coding for a cis-prenyltransferase(CPT) family protein, the gene coding for a Nogo-B receptor (NgBR)family protein, and the gene coding for a rubber elongation factor (REF)family protein is derived from a plant.
 16. The transformed plantaccording to claim 15, wherein at least one selected from the groupconsisting of the gene coding for a cis-prenyltransferase (CPT) familyprotein, the gene coding for a Nogo-B receptor (NgBR) family protein,and the gene coding for a rubber elongation factor (REF) family proteinis derived from Hevea brasiliensis.
 17. The transformed plant accordingto claim 14, wherein the gene coding for a cis-prenyltransferase (CPT)family protein is the following DNA [1] or [2]: [1] a DNA comprising thenucleotide sequence of SEQ ID NO:1; or [2] a DNA that hybridizes understringent conditions with a DNA comprising a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO:1, and codes for aprotein having an enzyme activity that catalyzes a reaction of cis-chainelongation of an isoprenoid compound.
 18. The transformed plantaccording to claim 14, wherein the gene coding for a Nogo-B receptor(NgBR) family protein is the following DNA [3] or [4]: [3] a DNAcomprising the nucleotide sequence of SEQ ID NO:3; or [4] a DNA thathybridizes under stringent conditions with a DNA comprising a nucleotidesequence complementary to the nucleotide sequence of SEQ ID NO:3, andcodes for a protein having the functions of binding to a membrane viaone or more transmembrane domains on the N-terminal side of the protein,and interacting with another protein on the C-terminal side thereof. 19.The transformed plant according to claim 14, wherein the gene coding fora rubber elongation factor (REF) family protein is the following DNA [5]or [6]: [5] a DNA comprising the nucleotide sequence of SEQ ID NO:5; or[6] a DNA that hybridizes under stringent conditions with a DNAcomprising a nucleotide sequence complementary to the nucleotidesequence of SEQ ID NO:5, and codes for a rubber particle-associatedprotein that is bound to rubber particles in latex.
 20. A method forproducing a pneumatic tire, the method comprising the steps of: kneadinga polyisoprenoid produced by the method according to claim 1 with anadditive to obtain a kneaded mixture; building a green tire from thekneaded mixture; and vulcanizing the green tire.
 21. A method forproducing a rubber product, the method comprising the steps of: kneadinga polyisoprenoid produced by the method according to claim 1 with anadditive to obtain a kneaded mixture; forming a raw rubber product fromthe kneaded mixture; and vulcanizing the raw rubber product.
 22. Amethod for producing a pneumatic tire, the method comprising the stepsof: kneading a polyisoprenoid produced by the method according to claim8 with an additive to obtain a kneaded mixture; building a green tirefrom the kneaded mixture; and vulcanizing the green tire.
 23. A methodfor producing a rubber product, the method comprising the steps of:kneading a polyisoprenoid produced by the method according to claim 8with an additive to obtain a kneaded mixture; forming a raw rubberproduct from the kneaded mixture; and vulcanizing the raw rubberproduct.